Patent Publication Number: US-2023136331-A1

Title: Immunocytokine containing il-21r mutein

Description:
1. CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63/250,911 filed on Sep. 30, 2021 and 63/351,298 filed on Jun. 10, 2022, the entire contents of which are incorporated by reference herein. 
    
    
     2. SEQUENCE LISTING 
     The instant application contains a sequence listing with 240 sequences which has been submitted via USPTO Patent Center is hereby incorporated by reference in its entirety. Said XML copy, created Sep. 29, 2022, is named “50042US_015US_CRF_sequencelisting.xml” and is 301,157 bytes in size. 
     3. BACKGROUND OF THE INVENTION 
     For the last decade, immune checkpoint blockade (ICB) represented by anti-PD-1 or anti-CTLA-4 antibody has led to considerable success in cancer immunotherapy, in which ICBs reprogram the immune system of patients to be against cancer. Despite the outstanding effectiveness of these types of therapeutics, few patients have benefitted from ICBs because the most patients failed to develop durable immune responses and stop the progression of cancer growth. The long-lasting and durable effector function of activated T cells is essential for eliminating cancer cells from our body through T cell-mediated immune response. In chronic infection and cancer, most of the T cells exposed to persistent antigens followed by continuous T cell receptor stimulation are exhausted. The exhausted T cells in the tumor microenvironment show dysfunction of cytokine releases like IFN-γ and TNF-α, which is their major effector function and loss of proliferation capacity. Exhausted T cells are distinguished from effector and memory T cells by high level expression of co-inhibitory receptors such as PD-1, TIM-3, or CTLA-4 on their surface. Another noticeable feature of fully differentiated exhausted T cells is epigenetic stability which might be the main reason for the resistance to ICB treatment. 
     A recent study done by Kristen E. Pauken reported that the epigenetic fate inflexibility of the genome of exhausted T cells impedes the transition of exhausted T cell into memory T cell, which is expected to be triggered by ICB treatment. This suggests that epigenetic reprogramming of exhausted T cells into memory T cells which have the potential for self-renewal and durable effector function, might be a solution for the limitation of current cancer immunotherapeutic. 
     Epigenetic reprogramming is accompanied by changes in the expression level of writer enzymes such as histone methyl transferases (HMT), histone acetyl transferases (HAT), or DNA methyltransferase (DNMT), all of which can alter the chromatin states determining the expression or suppression of a gene. It is well known that the signal triggered by a cytokine in immune cells regulates the expression level or activity of writer enzymes, which determines the differentiation fate of immune cells. From all types of cytokines, gamma chain cytokines, namely IL-2, IL-4, IL-7, IL-9, and IL-21, are known that have prominent roles in the activation of effector T cells or differentiation of memory T cells, suggesting that they can be potential candidates for anti-cancer immunotherapeutic. These cytokines can cause changes in chromosome accessibility and chromatin structure by altering the expression level of several transcription factors responsible for epigenetic modification. For example, TCF-1, a transcription factor expressed in T cells, is known that has intrinsic HDAC (histone deacetylase) activity and regulates gene expression by modifying chromatin accessibility. It was reported that the expression of TCF-1 in T cells can be induced by the treatment of cytokines like IL-7, IL-15, or IL-21 in vitro culture or in vivo experiment. Recently, lineage tracing based on single-cell sequencing analysis elucidated that TCF-1 is a key biomarker for progenitor exhausted CD4+ or CD8+ T cells (TPEX) respond to ICB treatment. This means that manipulating the expression of transcription factors like TCF-1 induced by cytokine in T cells can be another option for cancer immunotherapy. For several decades, there have been attempts to use these cytokines for cancer immunotherapy. 
     However, the clinical utility is minimal because of severe dose-limiting toxicities, leading a patient to death. In general, the expression of a cytokine receptor is ubiquitous all over the body, and the treatment of high doses of cytokine is related to systemic toxicities. Therefore, enhancing the specificity of a cytokine to increase the tolerable dose for systemic administration is required to solve toxicity-relating problems. 
     4. SUMMARY OF THE INVENTION 
     The present disclosure provides a novel immunocytokine specific to a target cell. The immunocytokine has activity specific to target cells by comprising a cytokine molecule (IL-21) fused to antigen binding protein (ABP) specific to a target protein and a capping moiety, interfering nonspecific binding of the cytokine molecule to a non-target cell. As a capping moiety, the present disclosure provides IL-21Rα mutein that has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     This immunocytokine binds to a target protein expressed on the surface of a certain cell type (e.g., immune cells) through its ABP, which results in accumulation of a cytokine close to the target cell. If a cytokine of the immunocytokine randomly binds to non-target cells before reaching to its target cell, a high dose of the cytokine might induce various side effects, and it may cause a narrow therapeutic index of the immunocytokine. To avoid this problem, the extracellular domain of IL-21Rα is used as a capping moiety to interfere with the binding of IL-21 to endogenous IL-21Rα (e.g., wild type IL21Rα (IL21RαWT)) on non-target cells. Since non-target cells lack a target protein that the ABP can recognize, the immunocytokine is not targeted to non-target cells and IL-21 stays capped by the capping moiety. Once immunocytokine with the capped IL-21 is delivered to a target cell, the capping moiety, the extracellular domain of IL-21Rα, is stripped off by competition with the endogenous IL21Rα (e.g., IL21RαWT) of a target cell, which can make IL-21 bind to the endogenous IL21Rα and transduce a signal to the target cell. 
     Since high binding affinity of IL-21 (approximately K D =50 pmol) to the extracellular domain of IL-21Rα can interfere with the competition between the extracellular domain of IL-21Rα of the immunocytokine and endogenous IL-21Rα of target cells, an extracellular domain of IL-21Rα in the immunocytokine was mutated (IL-21RαMutein) to have a lower binding affinity to IL-21. ABP of the immunocytokine can guide the complex comprising IL-21 and IL21RαMutein to specific target cells and the IL-21 brought to the target cells can bind and transduce signal to the target cells by competition between IL-21Rα mutein of the immunocytokine and endogenous IL-21 receptors on the surface of target cells. 
     Accordingly, the present disclosure provides: an immunocytokine, comprising:
         a. an antigen binding protein (ABP) specific to a target protein;   b. an IL-21 domain; and   c. an IL-21Rα mutein,
 
wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.
       

     In some embodiments, the target protein is an immune checkpoint molecule. In some embodiments, the target protein is PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, or CD20. 
     In some embodiments, the ABP is an antibody against the target protein. In some embodiments, the ABP is an immune check point inhibitor. In some embodiments, the ABP is anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is IgG. 
     In some embodiments, the ABP comprises Fc fragment selected from a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, and a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the sequence selected from SEQ ID NOs: 16, 185-190. 
     In some embodiments, the ABP comprises an Fc fragment with two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the first of the two Fc moieties, and the IL-21 domain is linked to the second of the two Fc moieties. In some embodiments, the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker or without a peptide linker. In some embodiments, the non-cleavable peptide linker is G4S linker having the sequence of SEQ ID NO: 17. In some embodiments, the non-cleavable peptide linker has a sequence selected from SEQ ID NOs: 212-224. 
     In some embodiments, the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof. In some embodiments, the ABP comprises V H  CDR1, V H  CDR2, V H  CDR3, V L  CDR1, V L  CDR2, and V L  CDR3 sequences of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the ABP comprises heavy chain and/or light chain of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain and/or a light chain variable domain of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the heavy chain variable domain and/or the light chain domain are linked to a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, or a human IgG4 Fc fragment. In some embodiments, the Fc fragment includes a mutation for knob-in-hole interaction, 
     In some embodiments, the ABP comprises:
         a. a heavy chain having the sequence of SEQ ID NO: 1 and a light chain having the sequence of SEQ ID NO: 2;   b. a heavy chain having the sequence of SEQ ID NO: 3 and a light chain having the sequence of SEQ ID NO: 4;   c. a heavy chain having the sequence of SEQ ID NO: 5 and a light chain having the sequence of SEQ ID NO: 6;   d. a heavy chain having the sequence of SEQ ID NO: 7 and a light chain having the sequence of SEQ ID NO: 8;   e. a heavy chain having the sequence of SEQ ID NO: 9 and a light chain having the sequence of SEQ ID NO: 10;   f. a heavy chain having the sequence of SEQ ID NO: 11 and a light chain having the sequence of SEQ ID NO: 12;   g. a heavy chain having the sequence of SEQ ID NO: 13 and a light chain having the sequence of SEQ ID NO: 14;   h. a heavy chain having the sequence of SEQ ID NO: 151 and a light chain having the sequence of SEQ ID NO: 152;   i. a heavy chain having the sequence of SEQ ID NO: 153 and a light chain having the sequence of SEQ ID NO: 154;   j. a heavy chain having the sequence of SEQ ID NO: 225 and a light chain having the sequence of SEQ ID NO: 226; or   k. a heavy chain having the sequence of SEQ ID NO: 227 and a light chain having the sequence of SEQ ID NO: 228.       

     In some embodiments, the ABP comprises:
         a. a heavy chain having the sequence of SEQ ID NO: 1 or a variation thereof, and a light chain having the sequence of SEQ ID NO: 2, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 1;   b. a heavy chain having the sequence of SEQ ID NO: 3 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 4, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 3;   c. a heavy chain having the sequence of SEQ ID NO: 5 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 6, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 5;   d. a heavy chain having the sequence of SEQ ID NO: 7 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 8, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 7;   e. a heavy chain having the sequence of SEQ ID NO: 9 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 10, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 9;   f. a heavy chain having the sequence of SEQ ID NO: 11 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 12, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 11;   g. a heavy chain having the sequence of SEQ ID NO: 13 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 14, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 13;   h. a heavy chain having the sequence of SEQ ID NO: 151 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 152, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 151;   i. a heavy chain having the sequence of SEQ ID NO: 153 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 154, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 153;   j. a heavy chain having the sequence of SEQ ID NO: 225 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 226, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 225; or   k. a heavy chain having the sequence of SEQ ID NO: 227 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 228, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 227.       

     In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has a sequence with at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). 
     In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence. 
     In some embodiments, the amino acid substitutions are selected from:
         a. Y10A   b. Q35K, Q35R, or Q35Y;   c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   d. E38A, E38C, E38K, E38R, or E38Y;   e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   f. F67A;   g. H68A;   h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;   l. I74A, I74H, I74K, I74R, or I74W;   m. L94A, L94F, L94K, L94Q, L94R, or L94Y;   n. P126A;   o. Y129A;   p. M130A;   q. K134A;   r. 5189A;   s. 5190A; and   t. Y191A.       

     In some embodiments, the amino acid substitutions are selected from:
         a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   b. E38C, E38R, or E38Y;   c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   g. D73A   h. I74R, or I74W; and   i. L94A, L94F, L94K, L94Q, L94R, or L94Y.       

     In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. 
     In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:
         a. a Fc fragment of a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, and 185-190; and   b. an IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169.       

     In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:
         a. a Fc fragment of a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, and 185-190;   b. a peptide linker; and   c. an IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169.       

     In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:
         a. a heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225, 227, or a variant thereof; and   b. an IL-21Rα mutein.       

     In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:
         a. a heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225, 227, or a variant thereof;   b. a peptide linker; and   c. an IL-21Rα mutein.       

     In some embodiments, the heavy chain of the ABP comprises a knob variant or a hole variant for knobs-in-holes interaction, wherein the knob variant and the hole variant comprise one or more modifications for the knobs-in-holes interaction. 
     In some embodiments, the heavy chain of the ABP comprises a variant of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227, wherein the variant has deletion of Lys (K) at the C-terminal end of the sequence. 
     In some embodiments, the heavy chain of the ABP comprises the sequence of SEQ ID NO: 103. 
     In some embodiments, the peptide linker is a G45 linker having the sequence of SEQ ID NO: 17. In some embodiments, the peptide linker has a sequence selected from SEQ ID NOs: 212-224. 
     In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the first chain has a sequence selected from SEQ ID NOs: 104-150 and 192-209. 
     In some embodiments, the immunocytokine comprises a second chain comprising a heavy chain of the ABP, a peptide linker and the IL-21 domain. In some embodiments, the heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the heavy chain of the ABP comprises a variant of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. The variant comprises deletion of lysine (Lys or K) at the C-terminal end of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the heavy chain of the ABP may comprise a knob variant or a hole variant for knobs-in-holes interaction. In some embodiments, the peptide linker is selected from SEQ ID NO: 17 and SEQ ID NOs: 212-224. In some embodiments, the second chain has the sequence of SEQ ID NO: 101. In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21). 
     In some embodiments, the immunocytokine comprises a first heavy chain and a second heavy chain of the ABP. In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation for knob-and-hole interaction. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation for knob-and-hole interaction. In some embodiments, the heavy chain is full length heavy chain or the fragment thereof. In some embodiments, the hole mutation and knob mutation are comprised in a Fc moiety of each heavy chain. In some embodiments, the hole mutation and knob mutation are comprised in a CH3 domain of each heavy chain. 
     In some embodiments, the immunocytokine comprises a light chain having the sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 102, 152, 154, 226 and 228. 
     In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21). 
     In another aspect, the present disclosure provides one or more polynucleotides encoding the immunocytokine provided herein. 
     In some embodiments, the one or more polynucleotides comprise:
         a. a first polynucleotide segment encoding a first chain comprising the heavy chain of the ABP and the IL-21Rα mutein;   b. a second polynucleotide segment encoding a second chain comprising the heavy chain of the ABP and the IL-21 domain; and   c. a third polynucleotide segment encoding the light chain of the ABP.       

     In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in a single polynucleotide molecule. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in multiple polynucleotide molecules. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate polynucleotide molecules. 
     In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides described herein. 
     In yet another aspect, the present disclosure provides a host cell comprising the one or more polynucleotides or the one or more vectors described herein. In some embodiments, host cell comprises the immunocytokine provided herein. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell. 
     The host cell can be a eukaryotic cell, for example a fungal cell such as yeast. The host cell can be a mammalian cell (which may be a cell in cell culture, or a cell present in a tissue or organ). In some embodiments, the host cell is a human, mouse, rat, rabbit, bovine or dog (or, for example, any other wild, livestock/domesticated animal) cell. In some embodiments, the host cell is a stable cell line cell, or a primary cell, adherent or suspension cell. As examples, the host cell can be a macrophage, osteosarcoma, or CHO, BHK (baby hamster kidney), Bowes human melanoma cell, 911, AT1080, A549, HEK293, or HeLa cell line cell or a mouse primary cell, but not limited thereto. In some embodiments, the host cell is a bacterial cell, such as  E. coli.    
     The eukaryotic cell can be a plant cell (for example a monocotyledonous or dicotyledonous plant cell; typically an experimental, crop and/or ornamental plant cell, for example  Arabidopsis,  maize); fish (for example Zebra fish; salmon), bird (for example chicken or other domesticated bird), insect (for example  Drosophila;  bees),  Nematoidia  or  Protista  (for example  Plasmodium  spp or  Acantamoeba  spp) cell. 
     In one aspect, the present disclosure provides a method of enhancing immune response in a subject, comprising administration of the immunocytokine described herein or the host cell described herein to the subject. In some embodiments, the subject is a cancer patient. 
     In one aspect, the present disclosure provides a method of selectively activating an IL-21Rα on a target cell, comprising: delivering the immunocytokine of the present disclosure to the target cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell. 
     Another aspect of the present disclosure provides an IL-21Rα mutein having a reduced binding affinity to an IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the wild-type IL-21Rα comprises the sequence of SEQ ID NO: 15. In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100. 
     In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has a sequence with at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). 
     In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). 
     In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence. 
     In some embodiments, the amino acid substitutions are selected from:
         a. Y10A   b. Q35K, Q35R, or Q35Y;   c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   d. E38A, E38C, E38K, E38R, or E38Y;   e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   f. F67A;   g. H68A;   h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;   l. I74A, I74H, I74K, I74R, or I74W;   m. L94A, L94F, L94K, L94Q, L94R, or L94Y;   n. P126A;   o. Y129A;   p. M130A;   q. K134A;   r. S189A;   s. S190A; and   t. Y191A.       

     In some embodiments, the amino acid substitutions are selected from:
         a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   b. E38C, E38R, or E38Y;   c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   g. D73A   h. I74R, or I74W; and   i. L94A, L94F, L94K, L94Q, L94R, or L94Y.       

     In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. 
     In another aspect, the present disclosure provides a polynucleotide comprising a coding sequence of the IL-21 Rα mutein described herein. In yet another aspect, the present disclosure provides a vector comprising the polynucleotide. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a recombinant AAV or lentiviral vector. 
     The present disclosure also provides a host cell comprising the IL-21 Rα mutein, the polynucleotide, or the vector described herein. 
    
    
     
       5. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: 
         FIG.  1    provides a schematic representation of an exemplary immunocytokine (αPD-1IL21RαMutein/IL21). 
         FIGS.  2 A- 2 V  provide sensorgrams from SPR full kinetics assay of IL-21Rα muteins against IL21. 
         FIG.  3    provides experimental results testing 66 different αPD-1IL21RαMutein/IL21. X-axis shows the affinity of immunocytokines to IL21 measured by Bio Layer interferometry (BLI) and y-axis shows efficacy of IL-21 mediated STAT3 phosphorylation (efficacy coefficient). The results show that as IL21Rα mutein in the immunocytokine has a reduced binding affinity to IL-21, the immunocytokine (αPD-1IL21RαMutein/IL21) has a higher efficacy coefficient. 
         FIG.  4    provides concentration dependent curves of selected six αPD-1IL21RαMutein/IL21, αPD-1IL21RαWT/IL21 (“WT”) and recombinant human IL-21 protein (“IL-21”). Specifically, the graph shows IL-21 mediated activation (efficacy coefficient) in PD-1(+) H9 cells. The max potency of αPD-1IL21RαMutein/IL21 with M70Q and M70H mutation was comparable to the recombinant human IL-21 (“IL-21”), and the others showed at least above 80%. 
         FIG.  5    provides concentration dependent curves of selected six αPD-1IL21RαMutein/IL21, αPD-1IL21RαWT/IL21 (“WT”) and recombinant human IL-21 protein (“IL-21”). Specifically, the graph shows IL-21 mediated activation (efficacy coefficient) in PD-1(−) H9 cells. The max potency of six αPD-1IL21RαMutein/IL21 was similar to the recombinant human IL-21 (“IL-21”), but EC50 increased in all variants. 
         FIGS.  6 A- 6 E  provide response curve of STAT3 phosphorylation observed in PD-1(−) H9 cells and PD-1(+) H9 cells in response to 16 variants of αPD-1IL21RαMutein/IL21. 
         FIGS.  7 A- 7 Q  provide sensorgrams from SPR full kinetics assay of immunocytokines (αPD-1IL21RαMutein/IL21) against PD-1. 
         FIG.  8 A  shows measurements of the binding affinities of the αPD-1 antibody or Immunocytokine to FcRn using ForteBio Octet RED96e instruments.  FIG.  8 B  is a table summarizing binding kinetics of the αPD-1 antibody or Immunocytokine to FcRn. 
         FIGS.  9 A,  9 B and  9 C  provide sensorgrams data from SPR full kinetics assay of immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) against their targets (hCTLA-4, hTIGIT, or hLAG-3). 
         FIG.  10    shows IFNγ concentrations (pg/ml) released from CTLs (effector cells) co-cultured with immunocytokines (αPD-1IL21RαMutein/IL21 including M70D, M70Q, L94K and E38R) as described in Section 5.7. The data are compared against IFNγ release in response to αPD-1 antibody or αPD-1IL21RαWT/IL21 (“WT”). 
         FIGS.  11 A and  11 B  show fluorescent signals (RFU) of Calcein AM released from dead tumor cells as described in Section 5.7.2. The signal indicates tumor killing efficacy of effector cells against tumor cells (MeWo cell line ( FIG.  11 A ) and A375_CMV cell line ( FIG.  11 B )) treated with immunocytokines (αPD-1IL21RαMutein/IL21 including M70D, M70Q, L94K and E38R) or controls (αPD-1 antibody or αPD-1IL21RαWT/IL21 (“WT”)). 
         FIG.  12    provides STAT3 phosphorylation curves obtained from HTRF-based high-throughput assay described in Section 5.9. It shows STAT3 phosphorylation induced by rhIL21, ABP-IL21RαWT/IL21, and ABP-IL21RαMutein/IL21, but not by antibodies without IL21 conjugation (i.e., anti-CTLA-4 antibody (Ipilimumab), anti-TIGIT antibody (Tiragolumab), anti-LAG-3 antibody (Relatlimab)). 
     
    
    
     6. DETAILED DESCRIPTION OF THE INVENTION 
     6.1. Definitions 
     The term “IL-21Rα mutein”, “IL-21RαMutein”, “IL21Rα mutein” or “IL21RαMutein” as used herein refers to the ectodomain of an IL-21Rα having one or more modifications. The modifications can be amino acid substitution, insertion, deletion or other mutation. In some embodiments, IL-21Rα mutein includes one or more biological, chemical, or both modifications compared to wild-type human IL-21Rα or its ectodomain. In some embodiments, the ectodomain of the wild-type human IL-21Rα comprises the sequence of SEQ ID NO: 15. 
     The term “pharmaceutically acceptable carrier” as used herein refers to a carrier or diluent that does not impair the biological activity and characteristics of an immunocytokine according to the present invention. As a pharmaceutically acceptable carrier in a composition that is formulated as a liquid solution, a sterile and biocompatible carrier can be used. The pharmaceutically acceptable carrier can be physiological saline, sterile water, Ringer&#39;s solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more thereof. In addition, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Further, the composition of the present invention can be formulated as injectable forms such as aqueous solutions, suspensions or emulsions with the aid of diluents, dispersants, surfactants, binders and lubricants. In addition, the composition according to the present invention can be formulated in the form of pills, capsules, granules, or tablets. Other carriers known in the art, e.g., as described in a literature [Remington&#39;s Pharmaceutical Sciences (E. W. Martin)], can be used. 
     The term “antigen-binding protein (ABP)” refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, the ABP binds the extracellular domain of the target protein. In certain embodiments, the ABP provided herein binds to an epitope of the target protein that is conserved between or among various species. 
     In some embodiments, the ABP is an antibody and the antibody can be a monoclonal antibody, a polyclonal antibody, a multi-specific antibody, a dual-specific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody. In some embodiments, the ABP comprises one or more heavy chain or a fragment thereof. In some embodiments, the ABP comprises one or more light chain or a fragment thereof. In some embodiments, the antibody comprises two heavy chains and two light chains, or fragments thereof. In some embodiments, the fragment of the heavy chain comprises Fc fragment, CH3 domain, or CH2 domain of the heavy chain. 
     The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), diabody, gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD 3  (e.g., Tetranectins), Fynomers, and LDLR-A module (e.g., Avimers). Additional information on alternative scaffolds is provided in Binz et al.,  Nat. Biotechnol.,  2005 23:1257-1268; Skerra,  Current Opin. In Biotech.,  2007 18:295-304; and Silacci et al.,  J. Biol. Chem.,  2014, 289:14392-14398; each of which is incorporated by reference in its entirety. An alternative scaffold is one type of ABP. 
     The term “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, antigen-binding fragments (Fab), F(ab′) 2  fragments, Fab′ fragments, single chain variable fragments (scFv, sFv), scFv-Fc fragments. Disulfide-linked Fv fragments, and a single domain antibody (sdAb). 
     The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope. 
     The term “Fe fragment” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini,  J. Allergy Clin. Immunol.,  2010, 125: S41-52, incorporated by reference in its entirety. The Fc fragment can comprise two Fc moieties. The Fc moiety can comprise a CH2-CH3 domain of a heavy chain. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein each Fc moiety is independently selected from IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. In some embodiments, the ABP comprises two Fc moieties of IgG1. In some embodiments, the ABP comprises two Fc moieties of IgG4. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein the first Fc moiety is an Fc moiety of IgG1 and the second Fc moiety is an Fc moiety of IgG4. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein the first Fc moiety comprises an C H 3 of IgG1 and the second Fc moiety comprises an C H 3 of IgG4. 
     The Fc region may be a naturally occurring Fc region, or an Fc region modified as described elsewhere in this disclosure. For example, the Fc moiety can be a knob variant or a hole variant for knobs-in-holes interaction. The Fc fragment can comprise a knob variant and a hole variant of a C-terminal region of an immunoglobulin heavy chain. 
     In some cases, the Fc fragment is engineered to introduce mutations to reduce effector function of immunoglobulin, which minimize ADCC by reducing the binding affinity for FcγR. Those mutations are the so-called LALA mutation(L234A/L235A) for human IgG1 type and SPLE mutation (S228P/L235E). (see, e.g., Hezareh et al. J. Virol. (2001) 75(24): 12161-8). In further embodiments, the LALA or SPLE mutations are present in the Fc fragment with the knobs-into-holes mutations. 
     The Fc fragment can comprise the M252Y/S254T/T256E (“YTE”) mutations. The YTE mutations allow the simultaneous modulation of serum half-life, tissue distribution and activity of IgG 1  (see DalFAcqua et al., J Biol Chem. (2006) 281:23514-24; and Robbie et al., Antimicr oh Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutations are present in the antibody with the knobs-into-holes mutations. 
     The V H  and V L  regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each V H  and V L  generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al.,  Sequences of Proteins of Immunological Interest  5 th  ed. (1991) Public Health Service, National Institutes of Health, Bethesda, Md., incorporated by reference in its entirety. 
     The light chain from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of its constant domain. 
     The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. 
     The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997,  J. Mol. Biol.,  273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996,  J. Mol. Biol.  262:732-745 (“Contact” numbering scheme); Lefranc et al.,  Dev. Comp. Immunol.,  2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun,  J. Mol. Biol.,  2001, 309:657-70 (“Aho” numbering scheme); each of which is incorporated by reference in its entirety. 
     Table 1 provides exemplary positions of CDR1-L (CDR1 of V L ), CDR2-L (CDR2 of V L ), CDR3-L (CDR3 of V L ), CDR1-H (CDR1 of V H ), CDR2-H (CDR2 of V H ), and CDR3-H (CDR3 of V H ), as identified by the Kabat and Chothia schemes. For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes. 
     CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at www.bioinf.org.uk/abs/abnum/, and described in Abhinandan and Martin,  Immunology,  2008, 45:3832-3839 or bioinf.org.uk—Prof. Andrew C. R. Martin&#39;s group at UCL, incorporated by reference in its entirety. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Exemplary CDR residues according to 
               
               
                 Kabat and Chothia numbering schemes. 
               
            
           
           
               
               
               
               
            
               
                   
                 CDR 
                 Kabat 
                 Chothia 
               
               
                   
                   
               
               
                   
                 CDR1-L 
                 24-34 
                 24-34 
               
               
                   
                 CDR2-L 
                 50-56 
                 50-56 
               
               
                   
                 CDR3-L 
                 89-97 
                 89-97 
               
               
                   
                 CDR1-H (Kabat Numbering) 
                     31-35B 
                 26-32 or 34* 
               
               
                   
                 CDR1-H (Chothia Numbering) 
                 31-35 
                 26-32 
               
               
                   
                 CDR2-H 
                 50-65 
                 52-56 
               
               
                   
                 CDR3-H 
                  95-102 
                  95-102 
               
               
                   
                   
               
               
                   
                 *The C-terminus of CDR1-H, when numbered using the Kabat numbering convention, varies between 32 and 34, depending on the length of the CDR. 
               
            
           
         
       
     
     The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminish of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. 
     6.2. Other Interpretational Conventions 
     Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. 
     Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof. 
     6.3. Summary of Experimental Observation 
     The present disclosure provides IL21RαMutein having a reduced affinity to IL-21 compared to IL21RαWT, and immunocytokines comprising the IL21RαMutein as a capping moiety. 
     As one example, the present disclosure provides an immunocytokine (αPD-1IL21RαMutein/IL21) comprising an ABP targeting PD-1. The immunocytokine can be targeted to PD-1 expressing cells, such as CD4+ or CD8 T+ cells. The immunocytokine comprises four polypeptide chains—two identical light chains and two different heavy chains—joined to form a heterodimer by knobs-into-holes (KiH) interaction. In the immunocytokine, one of the two heavy chains is fused to IL-21 and the other one is fused to a capping moiety, which is a mutant of ectodomain of IL-21Rα (IL-21RαMutein). IL-21 and the capping moiety are fused to the heavy chains through a non-cleavable and flexible polypeptide linker. 
     Applicant expressed the immunocytokines in CHO cells and purified them with a purity of ≥95%. The immunocytokine had 185 kDa molecular weight in the de-glycosylated form and 195 kDa in the glycosylated form when measured by mass spectrometry. Applicant also confirmed that over 90% of the molecules were present in the heterodimeric form of αPD-1IL21RαMutein/IL21. Applicant further measured activity of anti-PD-1 antibody using the PD-L1/TCR activator-CHO recombinant cell line (BPS bioscience) which can measure the intensity of TCR signaling through a luciferase reporter system driven by an NFAT-response element. The experiment showed that the fusion of IL21RαMutein/IL21 to IgG had little effect on the activity of anti-PD-1 antibody. Furthermore, SPR analysis demonstrated that the fusion of IL-21WT or IL21RαMutein and IL-21 to the anti-PD-1 antibody did not affect the affinity to PD-1 ( FIG.  7 A- 7 Q ). 
     Applicant generated 66 candidates of immunocytokine comprising anti-PD-1 IgG, IL-21, and one of various muteins of IL-21Rα. Next, using a high throughput HTRF assay, Applicant tested whether application of αPD-1IL21RαMutein/IL21 increases phosphorylation of STAT3 in PD-1(+) T cell. Based on the results from the HTRF assay, six αPD-1IL21RαMutein/IL21 candidates were selected. The selected candidates showed max potency at lower concentration compared to control αPD-1IL21RαWT/IL21 treatment and acted selectively on PD-1(+) cells. They showed superior potency at lower concentration compared to the control immunocytokine (αPD-1IL21RαWT/IL21). 
     Anti-cancer efficacy of the selected candidates can be tested in a humanized PDX mouse model. When αPD-1IL21RαMutein/IL21 binds to PD-1 expressed on PD-1(+) T cells, the reduced binding affinity of IL21RαMutein to human IL-21 can allow IL-21 of the immunocytokine to compete with and bind to endogenous IL21Rα (e.g., IL21RαWT), and lead to the invigoration of PD-1(+) T cells for the generation of durable anti-cancer immunity. 
     In summary, the present disclosure provides an immunocytokine that can exclusively deliver IL-21 to PD-1(+) T cells and reinvigorate the T cells to acquire a memory-like phenotype for long-lasting anti-cancer immunity. 
     6.4. IL-21Rα Muteins 
     In one aspect, the present disclosure provides IL-21Rα muteins having a reduced binding affinity to an IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has a mutation at the binding site of IL-21Rα against IL-21. In some embodiments, the IL-21Rα mutein has one or more amino acid substitution, insertion, or deletion at a binding site of IL-21Rα against IL-21. 
     In some embodiments, the IL-21Rα mutein specifically binds to the IL-21 domain, but with a reduced affinity. In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 50-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 200-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 300-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the IL-21Rα mutein has 10 to10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 2,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 1,000 to 2,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 2,000 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the IL-21Rα mutein has about 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 2500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     In some embodiments, the wild-type IL-21Rα is the ectodomain of a human IL-21Rα. In some embodiments, the wild-type IL-21Rα has the sequence of SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 96% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 97% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 98% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 99% sequence identity to SEQ ID NO: 15. 
     In some embodiments, the IL-21 Rα mutein includes one or more modifications at a binding site involved in the interaction between IL-21 and IL-21 Rα. In some embodiments, the one or more modifications are amino acid substitution, deletion, insertion, or a combination thereof. In some embodiments, the one or more modifications are chemical modifications. In some embodiments, the modifications can induce structural change in the binding site. 
     In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises one amino acid substitution compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises two amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises three amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises four amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises five amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises more than five amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15. 
     In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21Rα mutein comprises an amino acid substitution at one amino acid position selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. 
     In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21Rα mutein comprises an amino acid substitution at one amino acid position selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence. 
     In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94. 
     In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one amino acid position selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one amino acid position selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94. 
     In some embodiments, the one or more amino acid substitutions are selected from:
         a. Y10A   b. Q35K, Q35R, or Q35Y;   c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   d. E38A, E38C, E38K, E38R, or E38Y;   e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   f. F67A;   g. H68A;   h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;   l. I74A, I74H, I74K, I74R, or I74W;   m. L94A, L94F, L94K, L94Q, L94R, or L94Y;   n. P126A;   o. Y129A;   p. M130A;   q. K134A;   r. S189A;   s. S190A; and   t. Y191A.       

     In some embodiments, the one or more amino acid substitutions are selected from:
         a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;   b. E38C, E38R, or E38Y;   c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;   d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;   e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;   f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;   g. D73A   h. I74R, or I74W; and   i. L94A, L94F, L94K, L94Q, L94R, or L94Y.       

     In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the IL-21Rα mutein comprises a functional fragment of a protein having a sequence selected from SEQ ID NOs: 18-99 and 155-169. The functional fragment can bind to the IL-21 domain. 
     6.5. Immunocytokines 
     In another aspect, the present disclosure provides an immunocytokine comprising: (i) an antigen binding protein (ABP) specific to a target protein; (ii) an IL-21 domain; and (iii) an IL-21Rα mutein, wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. 
     The immunocytokine can comprise an IL-21Rα mutein disclosed in section 6.4. In some embodiments, the immunocytokine is one selected from R-kine-1 to 66. 
     6.5.1. Antigen Binding Protein (ABP) 
     The immunocytokine disclosed herein comprises an antigen binding protein (ABP) specific to a target protein. 
     The target protein can be a surface protein of an immune cell. In some embodiments, the target protein is a surface protein specific to a T cell. In some embodiments, the target protein is specific to CD4+ or CD8 T+ cells. 
     In some embodiments, the target protein is an immune checkpoint molecule. In some embodiments, the target protein is PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, or CD20. 
     In some embodiments, the ABP is an antibody against the target protein or a fragment thereof. 
     In some embodiments, the ABP is an immune check point inhibitor. In some embodiments, the ABP is anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is IgG. In some embodiments, the ABP is anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is IgG. In some embodiments, the ABP is anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is IgG. In some embodiments, the ABP is anti-LAG-3 antibody. In some embodiments, the anti-LAG-3 antibody is IgG. 
     In some embodiments, the ABP comprises Fc fragment selected from a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, and a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG1 Fc fragment. In some embodiments, the Fc fragment comprises a modification for knob-hole interaction. In some embodiments, the Fc fragment is engineered to introduce mutations to reduce effector function of immunoglobulin, which minimize ADCC by reducing the binding affinity for FcγR. In some embodiments, the Fc fragment comprises the sequence selected from SEQ ID NOs: 16, 185-190. In some embodiments, the Fc fragment is engineered to increase stability of the Fc fragment or the immunocytokine containing the Fc fragment. For example, the Fc fragment is engineered to remove Lys (K) at the C-terminal end. 
     In some embodiments, the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof. A functional variant refers to an ABP having one or more modification compared to an original ABP but maintaining the binding affinity and/or specificity of the original ABP. In some embodiments, the functional variant comprises a binding domain of the original ABP and a heterologous Fc fragment. 
     In some embodiments, the ABP comprises V H  CDR1, V H  CDR2, V H  CDR3, V L  CDR1, V L  CDR2, and V L  CDR3 sequences of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a light chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain and a light chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. 
     In some embodiments, the ABP comprises:
         a. a heavy chain having the sequence of SEQ ID NO: 1 and a light chain having the sequence of SEQ ID NO: 2;   b. a heavy chain having the sequence of SEQ ID NO: 3 and a light chain having the sequence of SEQ ID NO: 4;   c. a heavy chain having the sequence of SEQ ID NO: 5 and a light chain having the sequence of SEQ ID NO: 6;   d. a heavy chain having the sequence of SEQ ID NO: 7 and a light chain having the sequence of SEQ ID NO: 8;   e. a heavy chain having the sequence of SEQ ID NO: 9 and a light chain having the sequence of SEQ ID NO: 10;   f. a heavy chain having the sequence of SEQ ID NO: 11 and a light chain having the sequence of SEQ ID NO: 12;   g. a heavy chain having the sequence of SEQ ID NO: 13 and a light chain having the sequence of SEQ ID NO: 14;   h. a heavy chain having the sequence of SEQ ID NO: 151 and a light chain having the sequence of SEQ ID NO: 152;   i. a heavy chain having the sequence of SEQ ID NO: 153 and a light chain having the sequence of SEQ ID NO: 154;   j. a heavy chain having the sequence of SEQ ID NO: 225 and a light chain having the sequence of SEQ ID NO: 226; or   k. a heavy chain having the sequence of SEQ ID NO: 227 and a light chain having the sequence of SEQ ID NO: 228.       

     In some embodiments, the ABP comprises:
         a. a heavy chain having the sequence of SEQ ID NO: 1 or a variation thereof, and a light chain having the sequence of SEQ ID NO: 2, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 1;   b. a heavy chain having the sequence of SEQ ID NO: 3 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 4, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 3;   c. a heavy chain having the sequence of SEQ ID NO: 5 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 6, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 5;   d. a heavy chain having the sequence of SEQ ID NO: 7 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 8, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 7;   e. a heavy chain having the sequence of SEQ ID NO: 9 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 10, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 9;   f. a heavy chain having the sequence of SEQ ID NO: 11 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 12, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 11;   g. a heavy chain having the sequence of SEQ ID NO: 13 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 14, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 13;   h. a heavy chain having the sequence of SEQ ID NO: 151 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 152, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 151;   i. a heavy chain having the sequence of SEQ ID NO: 153 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 154, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 153;   j. a heavy chain having the sequence of SEQ ID NO: 225 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 226, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 225; or   k. a heavy chain having the sequence of SEQ ID NO: 227 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 228, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 227.       

     In preferred embodiments, the ABP comprises two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the first of the two Fc moieties, and the IL-21 domain is linked to the second of the two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the C terminus of the first of the two Fc moieties, and the IL-21 domain is linked to the C terminus of the second of the two Fc moieties. Various methods known in the art can be used to link the IL-21Rα mutein to the first of the two Fc moieties, and the IL-21 domain to the second of the two Fc moieties. In some embodiments, the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker or without a peptide linker. In some embodiments, the non-cleavable peptide linker is G4S linker having the sequence of SEQ ID NO: 17. In some embodiments, a non-peptide linker is used. In some embodiments, the non-cleavable peptide linker has a sequence selected from SEQ ID NOs: 212-224. 
     In some embodiments, the ABP comprises an Fc moiety of a human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc moiety comprises any one sequence selected from SEQ ID NOs: 16, and 185-190. In some embodiments, the Fc moiety comprises an C H 3 domain of a human IgG1, IgG2, IgG3 or IgG4. 
     In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP is a Fv fragment, a Fab fragment, a F(ab′) 2  fragment, a Fab′ fragment, a scFv (sFv) fragment, and a scFv-Fc fragment. 
     In some embodiments, the ABP comprises a knob variant and a hole variant of Fc fragment. 
     6.5.2. IL-21 Domain 
     In some embodiments, the IL-21 domain is a human IL-21. In some embodiments, the IL-21 domain is a functional fragment of human IL-21, which can bind to IL-21Rα and activate the target cell. In some embodiments, the IL-21 domain is a functional variant or a homolog of human IL-21, which can bind to IL-21Rα and activate the target cell. 
     In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21). In some embodiments, the IL-21 domain has a sequence at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 100. 
     6.5.3. Immunocytokine Structure 
     In some embodiments, the immunocytokine comprises four polypeptide chains-two identical light chains and two heavy chains, joined to form a heterodimer by knobs-into-holes (KiH) interaction. In some embodiments, one of the two heavy chains (“first chain”) is fused to a capping moiety (e.g., IL-21Rα mutein) and the other one (“second chain”) is fused to IL-21. In some embodiments, IL-21 and the capping moiety are fused to the heavy chains through a peptide linker. In some embodiments, the peptide linker is a non-cleavable and flexible peptide linker. 
     In some embodiments, the first chain comprising from the N terminus to C terminus:
         a. a first Fc moiety of the ABP; and   b. an IL-21Rα mutein.       

     In some embodiments, the first chain further comprises a linker between the first Fc moiety of the ABP and the IL-21Rα mutein. 
     In some embodiments, the first Fc moiety is a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, 185-190. In some embodiments, the first Fc moiety comprises an C H 3 domain of a human IgG1, IgG2, IgG3 or IgG4. 
     In some embodiments, the first chain comprising from the N terminus to C terminus:
         a. a first heavy chain of the ABP; and   b. an IL-21Rα mutein.       

     In some embodiments, the first chain further comprises a linker between the first heavy chain of the ABP and the IL-21Rα mutein. 
     In some embodiments, the first heavy chain of the ABP comprises a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227 or a variation thereof. In some embodiments, the variation comprises a knob-and-hole mutation in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the variation comprises removal of Lys (K) at the C-terminal end in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the variation comprises a knob-and-hole mutation and removal of Lys (K) at the C-terminal end in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. The knob-and-hole mutation can be a mutation for making a knob variant or for making a hole variant for knob-and-hole interaction. In some embodiments, the first heavy chain of the ABP comprises the sequence of SEQ ID NO: 103. 
     In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. 
     In some embodiments, the first chain comprises a sequence selected from SEQ ID NOs: 104-150 and 192-209. 
     In some embodiments, the first chain comprises a sequence selected from SEQ ID NOs: 170-184. 
     In some embodiments, the second chain comprises from the N terminus to C terminus:
         a. a second Fc moiety of the ABP; and   b. an IL-21 domain.       

     In some embodiments, the second chain further comprises a linker between the second Fc moiety of the ABP and the IL-21 domain. 
     In some embodiments, the second Fc moiety is a second heavy chain of the ABP. 
     In some embodiments, the immunocytokine comprises a first heavy chain and a second heavy chain of the ABP. In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation for knob-and-hole interaction. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation for knob-and-hole interaction. 
     In some embodiments, the second chain has the sequence of SEQ ID NO: 101. 
     In some embodiments, the immunocytokine comprises two identical light chains. In some embodiments, the light chain has the sequence of SEQ ID NO: 102. In some embodiments, the light chain is the light chain of any one of the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof. 
     6.6. Polynucleotide, Vector and Host Cells 
     One aspect of the present disclosure provides one or more polynucleotides encoding the immunocytokine. In some embodiments, the one or more polynucleotides comprise:
         a. a first polynucleotide segment encoding a first chain comprising the heavy chain of the ABP and the IL-21Rα mutein;   b. a second polynucleotide segment encoding a second chain comprising the heavy chain of the ABP and the IL-21 domain; and   c. a third polynucleotide segment encoding the light chain of the ABP.       

     In some embodiments, the first polynucleotide segment comprises a coding sequence of a first chain comprising the heavy chain of the ABP, a peptide linker and the IL-21Rα mutein. In some embodiments, the first polynucleotide segment comprises a coding sequence of a polypeptide having a sequence selected from SEQ ID NOs: 104-150 and 192-209. 
     In some embodiments, the second polynucleotide segment comprises a coding sequence of a second chain comprising the heavy chain of the ABP, a peptide linker and the IL-21 domain. In some embodiments, the second polynucleotide segment comprises a coding sequence of a polypeptide having the sequence of SEQ ID NO: 101. 
     In some embodiments, the third polynucleotide segment comprises a coding sequence of a light chain having the sequence of SEQ ID NO: 102. 
     In some embodiments, the first polynucleotide segment comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID NO: 210. In some embodiments, the first polynucleotide comprises a sequence of SEQ ID NO: 210 with one or more nucleotide differences corresponding to the one or more amino acid substitutions in IL21RαMutein. 
     In some embodiments, the first polynucleotide segment comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID NO: 211. In some embodiments, the first polynucleotide comprises a sequence of SEQ ID NO: 211 with one or more nucleotide differences corresponding to the one or more amino acid substitutions in IL21RαMutein. 
     In some embodiments, the one or more polynucleotides have a sequence which has been codon optimized for expression in a mammalian cell. In some embodiments, the one or more polynucleotides have a sequence which has been codon optimized for expression in a human cell. 
     In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in a single polynucleotide molecule. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in multiple polynucleotide molecules. 
     When more than one polynucleotide segments are present in a single polynucleotide molecule, the multiple polynucleotide segments can be separated by internal ribosome entry site (IRES). In some embodiments, the multiple polynucleotide segments are separated by a self-cleavage site. 
     In some embodiments, the one or more polynucleotides further comprise a regulatory sequence operably linked to the first, second, or third polynucleotide segment. In some embodiments, the one or more polynucleotides comprise more than one regulatory sequences. In some embodiments, the one or more polynucleotides comprise a regulatory sequence for each of the first, second and third polynucleotide segment. 
     In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate polynucleotide molecules. 
     In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate vectors. In some embodiments, two or more of the polynucleotide segments are cloned in a single vector. 
     In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector or a lentiviral vector. In some embodiments, the vector is non-viral. In some embodiments, the vector is a plasmid. 
     In some embodiments, the one or more polynucleotides or the one or more vectors are present in a host cell. Accordingly, one aspect of the present disclosure provides a host cell comprising the one or more polynucleotides or the one or more vectors. In some embodiments, the host cell expresses the immunocytokine. In some embodiments, the host cell comprises the immunocytokine. In some embodiments, the host cell releases the immunocytokine. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell. 
     The host cell can be a eukaryotic cell, for example a fungal cell such as yeast. The host cell can be a mammalian cell (which may be a cell in cell culture, or a cell present in a tissue or organ). In some embodiments, the host cell is a human, mouse, rat, rabbit, bovine or dog (or, for example, any other wild, livestock/domesticated animal) cell. In some embodiments, the host cell is a stable cell line cell, or a primary cell, adherent or suspension cell. As examples, the host cell can be a macrophage, osteosarcoma, or CHO, BHK (baby hamster kidney), Bowes human melanoma cell, 911, AT1080, A549, HEK293, or HeLa cell line cell or a mouse primary cell, but not limited thereto. In some embodiments, the host cell is a bacterial cell, such as  E. coli.    
     The eukaryotic cell can be a plant cell (for example a monocotyledonous or dicotyledenous plant cell; typically an experimental, crop and/or ornamental plant cell, for example  Arabidopsis,  maize); fish (for example Zebra fish; salmon), bird (for example chicken or other domesticated bird), insect (for example  Drosophila;  bees),  Nematoidia  or  Protista  (for example  Plasmodium  spp or  Acantamoeba  spp) cell. 
     In some embodiments, the host cell is used for production of the immunocytokine. In some embodiments, immunocytokine produced from the host cell is purified for therapeutic use. In some embodiments, the host cell is used as therapeutics. 
     One aspect of the present disclosure provides a polynucleotide encoding the IL-21Rα mutein. In some embodiments, the polynucleotide encoding IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the polynucleotide is a viral or non-viral vector. In some embodiments, the polynucleotide further comprises a regulatory sequence operable linked to the coding sequence of IL-21Rα mutein. In another aspect, the present disclosure provides a host cell comprising the polynucleotide encoding the IL-21Rα mutein. 
     6.7. Method of Treatment 
     In another aspect, the present disclosure provides a method of administering the immunocytokine or the host cell expressing immunocytokine described above to a subject. In some embodiments, the subject is a cancer patient. 
     In some embodiments, the administration is effective in enhancing immune response in the subject. In some embodiments, the administration is effective in treating cancer. In some embodiments, the administration is effective in selectively activating an IL-21Rα on a target cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell. 
     In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to enhance immune response in the subject. In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to treat cancer. In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to selectively activate an IL-21Rα on a target cell. 
     In some embodiments, the method comprises administration of the immunocytokine, the host cell or a pharmaceutical composition comprising the immunocytokine or the host cell. 
     6.8. Pharmaceutical Composition 
     In one aspect, the present disclosure provides a pharmaceutical composition comprising the immunocytokine or the host cell comprising the immunocytokine provided herein. 
     In some embodiments, the pharmaceutical composition comprises the immunocytokine and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising a host cell expressing the immunocytokine and a pharmaceutically acceptable carrier. 
     In some embodiments, the pharmaceutically acceptable carrier is a sterile aqueous solution or dispersion and sterile powder for preparation of a sterile injectable solution or dispersion. In some embodiments, the composition is formulated for parenteral injection. The composition can be formulated as a solid, a solution, a microemulsion, a liposome, or other ordered structures suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures thereof. In some cases, the composition contains an isotonic agent, for example, sugar, polyalcohol, for example, sorbitol or sodium chloride. 
     In some embodiments, the pharmaceutical composition is provided in a unit dose for use as described above. 
     7. EXAMPLES 
     7.1. Generation of IL21Rα Muteins 
     Nine (9) amino acid residues of IL-21Rα (M70, A71, D72, D73, Y36, E38, L39, I74, and L94) were predicted to form a binding site to IL-21 based on the predicted structure of IL-21 and IL-21Rα. Some amino acid residues (e.g., Q35) of IL-21Rα were additionally predicted to be involved in the binding affinity from the in-silico analysis (Discovery studio). Their roles in binding to IL-21 were further studied by alanine scanning mutagenesis of each of the amino acid residues of IL-21Rα. IL-21RαMuteins were designed by single amino acid substitution to the 20 amino acid residues in the IL-21Rα amino acid sequence as provided in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 WT and IL-21RαMuteins 
               
            
           
           
               
               
            
               
                 No. 
                 Point mutation 
               
               
                   
               
            
           
           
               
               
            
               
                 1 
                 WT 
               
               
                 2 
                 Y10A 
               
               
                 3 
                 Q35K 
               
               
                 4 
                 Q35R 
               
               
                 5 
                 Q35Y 
               
               
                 6 
                 Y36A 
               
               
                 7 
                 Y36C 
               
               
                 8 
                 Y36E 
               
               
                 9 
                 Y36G 
               
               
                 10 
                 Y36H 
               
               
                 11 
                 Y36I 
               
               
                 12 
                 Y36K 
               
               
                 13 
                 Y36M 
               
               
                 14 
                 Y36N 
               
               
                 15 
                 Y36P 
               
               
                 16 
                 Y36Q 
               
               
                 17 
                 Y36R 
               
               
                 18 
                 Y36S 
               
               
                 19 
                 Y36T 
               
               
                 20 
                 Y36V 
               
               
                 21 
                 E38A 
               
               
                 22 
                 E38C 
               
               
                 23 
                 E38K 
               
               
                 24 
                 E38R 
               
               
                 25 
                 E38Y 
               
               
                 26 
                 L39A 
               
               
                 27 
                 L39C 
               
               
                 28 
                 L39E 
               
               
                 29 
                 L39F 
               
               
                 30 
                 L39H 
               
               
                 31 
                 L39K 
               
               
                 32 
                 L39R 
               
               
                 33 
                 L39W 
               
               
                 34 
                 L39Y 
               
               
                 35 
                 F67A 
               
               
                 36 
                 H68A 
               
               
                 37 
                 M70C 
               
               
                 38 
                 M70D 
               
               
                 39 
                 M70F 
               
               
                 40 
                 M70G 
               
               
                 41 
                 M70H 
               
               
                 42 
                 M70K 
               
               
                 43 
                 M70L 
               
               
                 44 
                 M70N 
               
               
                 45 
                 M70Q 
               
               
                 46 
                 M70R 
               
               
                 47 
                 M70S 
               
               
                 48 
                 M70T 
               
               
                 49 
                 M70V 
               
               
                 50 
                 M70W 
               
               
                 51 
                 M70Y 
               
               
                 52 
                 A71E 
               
               
                 53 
                 A71F 
               
               
                 54 
                 A71I 
               
               
                 55 
                 A71L 
               
               
                 56 
                 A71Q 
               
               
                 57 
                 A71R 
               
               
                 58 
                 A71W 
               
               
                 59 
                 A71Y 
               
               
                 60 
                 D72A 
               
               
                 61 
                 D72C 
               
               
                 62 
                 D72E 
               
               
                 63 
                 D72G 
               
               
                 64 
                 D72H 
               
               
                 65 
                 D72K 
               
               
                 66 
                 D72Q 
               
               
                 67 
                 D72R 
               
               
                 68 
                 D72W 
               
               
                 69 
                 D72Y 
               
               
                 70 
                 D73A 
               
               
                 71 
                 I74A 
               
               
                 72 
                 I74K 
               
               
                 73 
                 I74R 
               
               
                 74 
                 I74W 
               
               
                 75 
                 L94A 
               
               
                 76 
                 L94F 
               
               
                 77 
                 L94K 
               
               
                 78 
                 L94Q 
               
               
                 79 
                 L94R 
               
               
                 80 
                 L94Y 
               
               
                 81 
                 P126A 
               
               
                 82 
                 Y129A 
               
               
                 83 
                 M130A 
               
               
                 84 
                 K134A 
               
               
                 85 
                 S189A 
               
               
                 86 
                 S190A 
               
               
                 87 
                 Y191A 
               
               
                   
               
            
           
         
       
     
     The IL21R muteins were generated by introducing one or more point mutations to a plasmid encoding wild type IL-21Rα. Human IgG1Fc (Pro100-Lys330) and IL21R α (Cys20-Glu232) wild type or muteins were conjugated by (G4S) 3  linker. Azurocidin signal peptide was added at the N-terminal for secretion of the expressed protein. After verification of the constructs by sequencing, a large-scale plasmid preparation was performed to obtain enough DNA for transfection. 
     7.2. SPR Full Kinetics Assay of IL-21Rα Muteins against IL-21 
     Bivalent Fc-fusion proteins (IgG1) were generated with each of the muteins [IL21RαMutein-Fc] and their binding affinity to IL-21 was measured by SPR (Biacore 8K) (Table 3 and  FIGS.  2 A- 2 V ). IL21Rα Muteins and IL-21&#39;s affinity was tested by CM5 sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CMS sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 1.55 ug/mL of hIL-21 in 10 mM NaAc (pH 5.0) to the channel for 240 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s. 
     Multiple cycle kinetics were used to perform the assay. hIL-21R (WT or Muteins) at 7 different concentrations and a running buffer were injected orderly to Fc1-Fc2 at a flow rate of 80 μL/min for an association phase of 120 s, followed by 1000 s dissociation. 10 mM glycine pH1.5 was injected as a regeneration buffer following every dissociation phase. 
     The sensorgrams from the reference channel Fc1 and the buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model or heterogeneous ligand. Molecular weight of 15 kDa were used to calculate the molar concentration of IL-21. 
     The data from the SPR full kinetics assay of IL-21Rα muteins against IL21 are provided in  FIGS.  2 A- 2 V . The binding affinities of muteins were measured using Biacore 8K and provided in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Binding affinity of Fc-IL21Rα (WT 
               
               
                 and mutein) against IL21 (1:1 binding model) 
               
            
           
           
               
               
               
            
               
                 No. 
                 Point Mutation 
                 K D  (M) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 WT 
                 1.67E−10 
               
               
                 2 
                 Y10A 
                 5.23E−10 
               
               
                 3 
                 Q35K 
                 3.43E−10 
               
               
                 4 
                 Q35R 
                 4.91E−10 
               
               
                 5 
                 Q35Y 
                 3.66E−10 
               
               
                 6 
                 Y36A 
                 2.13E−09 
               
               
                 7 
                 Y36C 
                 1.10E−09 
               
               
                 8 
                 Y36E 
                 6.88E−10 
               
               
                 9 
                 Y36G 
                 2.76E−09 
               
               
                 10 
                 Y36H 
                 6.11E−10 
               
               
                 11 
                 Y36I 
                 2.55E−09 
               
               
                 12 
                 Y36K 
                 2.97E−09 
               
               
                 13 
                 Y36M 
                 7.26E−10 
               
               
                 14 
                 Y36N 
                 8.94E−10 
               
               
                 15 
                 Y36P 
                 2.46E−07 
               
               
                 16 
                 Y36Q 
                 1.04E−09 
               
               
                 17 
                 Y36R 
                 6.67E−09 
               
               
                 18 
                 Y36S 
                 2.58E−09 
               
               
                 19 
                 Y36T 
                 3.98E−09 
               
               
                 20 
                 Y36V 
                 3.33E−09 
               
               
                 21 
                 E38A 
                 2.85E−08 
               
               
                 22 
                 E38C 
                 2.40E−08 
               
               
                 23 
                 E38K 
                 &gt;1.00E−06* 
               
               
                 24 
                 E38R 
                 &gt;1.00E−06  
               
               
                 25 
                 E38Y 
                 1.77E−07 
               
               
                 26 
                 L39A 
                 2.36E−08 
               
               
                 27 
                 L39C 
                 1.50E−07 
               
               
                 28 
                 L39E 
                 1.02E−07 
               
               
                 29 
                 L39F 
                 2.65E−10 
               
               
                 30 
                 L39H 
                 1.98E−09 
               
               
                 31 
                 L39K 
                 1.41E−08 
               
               
                 32 
                 L39R 
                 9.70E−08 
               
               
                 33 
                 L39W 
                 2.33E−09 
               
               
                 34 
                 L39Y 
                 8.16E−10 
               
               
                 35 
                 F67A 
                 4.37E−10 
               
               
                 36 
                 H68A 
                 1.68E−10 
               
               
                 37 
                 M70C 
                 &gt;1.00E−06  
               
               
                 38 
                 M70D 
                 &gt;1.00E−06  
               
               
                 39 
                 M70F 
                 1.18E−09 
               
               
                 40 
                 M70G 
                 &gt;1.00E−06  
               
               
                 41 
                 M70H 
                 6.94E−07 
               
               
                 42 
                 M70K 
                 &gt;1.00E−06  
               
               
                 43 
                 M70L 
                 6.42E−10 
               
               
                 44 
                 M70N 
                 1.16E−07 
               
               
                 45 
                 M70Q 
                 6.85E−07 
               
               
                 46 
                 M70R 
                 &gt;1.00E−06  
               
               
                 47 
                 M70S 
                 8.54E−08 
               
               
                 48 
                 M70T 
                 5.12E−09 
               
               
                 49 
                 M70V 
                 9.74E−10 
               
               
                 50 
                 M70W 
                 7.06E−07 
               
               
                 51 
                 M70Y 
                 7.92E−08 
               
               
                 52 
                 A71E 
                 2.92E−09 
               
               
                 53 
                 A71F 
                 1.01E−09 
               
               
                 54 
                 A71I 
                 1.97E−09 
               
               
                 55 
                 A71L 
                 1.26E−09 
               
               
                 56 
                 A71Q 
                 5.01E−09 
               
               
                 57 
                 A71R 
                 4.09E−07 
               
               
                 58 
                 A71W 
                 1.85E−08 
               
               
                 59 
                 A71Y 
                 1.03E−08 
               
               
                 60 
                 D72A 
                 &gt;1.00E−06  
               
               
                 61 
                 D72C 
                 &gt;1.00E−06  
               
               
                 62 
                 D72E 
                 1.14E−06 
               
               
                 63 
                 D72G 
                 &gt;1.00E−06  
               
               
                 64 
                 D72H 
                 &gt;1.00E−06  
               
               
                 65 
                 D72K 
                 &gt;1.00E−06  
               
               
                 66 
                 D72Q 
                 &gt;1.00E−06  
               
               
                 67 
                 D72R 
                 &gt;1.00E−06  
               
               
                 68 
                 D72W 
                 &gt;1.00E−06  
               
               
                 69 
                 D72Y 
                 &gt;1.00E−06  
               
               
                 70 
                 D73A 
                 &gt;1.00E−06  
               
               
                 71 
                 I74A 
                 7.66E−10 
               
               
                 72 
                 I74K 
                 5.18E−10 
               
               
                 73 
                 I74R 
                 1.30E−09 
               
               
                 74 
                 I74W 
                 1.15E−09 
               
               
                 75 
                 L94A 
                 1.44E−09 
               
               
                 76 
                 L94F 
                 1.49E−09 
               
               
                 77 
                 L94K 
                 2.79E−07 
               
               
                 78 
                 L94Q 
                 1.19E−09 
               
               
                 79 
                 L94R 
                 3.97E−08 
               
               
                 80 
                 L94Y 
                 1.13E−09 
               
               
                 81 
                 P126A 
                 2.77E−10 
               
               
                 82 
                 Y129A 
                 5.15E−10 
               
               
                 83 
                 M130A 
                 2.54E−10 
               
               
                 84 
                 K134A 
                 6.18E−10 
               
               
                 85 
                 S189A 
                 2.90E−10 
               
               
                 86 
                 S190A 
                 2.45E−10 
               
               
                 87 
                 Y191A 
                 6.67E−10 
               
               
                   
               
               
                 *1.00E−06 is the minimum detection limit. 
               
            
           
         
       
     
     After the measurement of binding affinities of the muteins [IL21Rα(mut)-Fc] to IL-21, 58 muteins were classified based on the degree of reduction in their binding affinity to IL-21, e.g., 10, 100, and 1000-fold reduction compared to wild-type IL-21Rα. Finally, 66 IgG-fusion proteins were generated in which an IL-21 and one of the muteins of IL21-Rα are fused to one of two heavy chains of IgG, respectively. 
     7.3. Generation of Immunocytokines (αPD-1IL21RαMutein/IL21) 
     The immunocytokine described herein, αPD-1IL21RαMutein/IL21, can exhibit an anti-cancer immune response by working as an ICB and inducing signal transduction mediated by the complex of IL21 receptor (IL21Rα/common gamma chain) expressed on the surface of target cells. αPD-1IL21RαMutein/IL21 is designed to primarily activate target immune cells only when it binds to PD-1. It leads competition between IL21RαMutein and endogenous IL21Rα (e.g., IL21RαWT) of target cells by the proximity, inducing stripping of IL21RαMutein from the moiety of IL-21, causing it to bind to endogenous IL21Rα. 
     Previously, a fusion protein comprising an attenuated IL-21 fused to the c-terminal ends of the anti-PD-1 antibody was developed for treatment of cancer by activation of immune cells. In the fusion protein, an attenuated IL-21 was used to reduce off-target effects and the anti-PD-1 antibody was used to improve bioavailability at the target. The attenuated IL-21 includes two point mutations in the amino acid sequence of IL-21, making its max potency reduced to 70-80% compared to wild-type IL-21 (See Shanling Shen et al. Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity. Front Immunol. 2020 May 8; 11:832). 
     Unlike the fusion protein comprising an attenuated IL-21, αPD-1IL21RαMutein/IL21 includes an unmodified IL-21, thus they can have effects on target cells similar to wild-type IL-21. 66 immunocytokines, each containing a different mutein of IL21Rα, were generated. The 66 immunocytokines (Table 4) include one or two amino acid substitutions. More specifically, the immunocytokines (R-kine-1 to 66) includes (i) a first chain comprising a heavy chain, G4S linker and IL-21RαMutein; (ii) a second chain comprising a heavy chain, G4S linker and a human IL-21; and (iii) two light chains, as specified in Table 4. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Sequence of first 
                 Sequence of second 
                   
               
               
                   
                 IL-21Rα 
                 chain (Heavy 
                 chain (Heavy 
               
               
                   
                 Mutation 
                 Chain-G4S Linker- 
                 Chain-G4S Linker- 
                 Sequence of two 
               
               
                 No. 
                 Site 
                 IL-21RαMutein) 
                 human IL-21) 
                 light chains 
               
               
                   
               
             
            
               
                 R-kine-1 
                 WT 
                 SEQ ID NO: 191 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-2 
                 Y36C 
                 SEQ ID NO: 104 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-3 
                 Y36E 
                 SEQ ID NO: 105 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-4 
                 Y36G 
                 SEQ ID NO: 106 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-5 
                 Y36H 
                 SEQ ID NO: 107 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-6 
                 Y36I 
                 SEQ ID NO: 108 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-7 
                 Y36K 
                 SEQ ID NO: 109 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-8 
                 Y36M 
                 SEQ ID NO: 110 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-9 
                 Y36N 
                 SEQ ID NO: 111 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-10 
                 Y36P 
                 SEQ ID NO: 112 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-11 
                 Y36Q 
                 SEQ ID NO: 113 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-12 
                 Y36R 
                 SEQ ID NO: 114 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-13 
                 Y36S 
                 SEQ ID NO: 115 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-14 
                 Y36T 
                 SEQ ID NO: 116 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-15 
                 Y36V 
                 SEQ ID NO: 117 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-16 
                 E38C 
                 SEQ ID NO: 118 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-17 
                 E38R 
                 SEQ ID NO: 200 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-18 
                 E38Y 
                 SEQ ID NO: 119 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-19 
                 L39C 
                 SEQ ID NO: 120 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-20 
                 L39E 
                 SEQ ID NO: 121 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-21 
                 L39H 
                 SEQ ID NO: 122 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-22 
                 L39K 
                 SEQ ID NO: 123 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-23 
                 L39R 
                 SEQ ID NO: 124 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-24 
                 L39W 
                 SEQ ID NO: 125 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-25 
                 L39Y 
                 SEQ ID NO: 126 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-26 
                 M70F 
                 SEQ ID NO: 127 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-27 
                 M70H 
                 SEQ ID NO: 128 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-28 
                 M70N 
                 SEQ ID NO: 129 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-29 
                 M70Q 
                 SEQ ID NO: 130 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-30 
                 M70S 
                 SEQ ID NO: 131 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-31 
                 M70T 
                 SEQ ID NO: 132 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-32 
                 M70V 
                 SEQ ID NO: 133 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-33 
                 M70W 
                 SEQ ID NO: 134 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-34 
                 M70Y 
                 SEQ ID NO: 135 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-35 
                 A71E 
                 SEQ ID NO: 136 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-36 
                 A71F 
                 SEQ ID NO: 137 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-37 
                 A71I 
                 SEQ ID NO: 138 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-38 
                 A71L 
                 SEQ ID NO: 139 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-39 
                 A71Q 
                 SEQ ID NO: 140 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-40 
                 A71R 
                 SEQ ID NO: 141 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-41 
                 A71W 
                 SEQ ID NO: 142 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-42 
                 A71Y 
                 SEQ ID NO: 143 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-43 
                 I74R 
                 SEQ ID NO: 144 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-44 
                 I74W 
                 SEQ ID NO: 145 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-45 
                 L94F 
                 SEQ ID NO: 146 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-46 
                 L94K 
                 SEQ ID NO: 147 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-47 
                 L94Q 
                 SEQ ID NO: 148 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-48 
                 L94R 
                 SEQ ID NO: 149 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-49 
                 L94Y 
                 SEQ ID NO: 150 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-50 
                 M70C 
                 SEQ ID NO: 201 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-51 
                 M70D 
                 SEQ ID NO: 202 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-52 
                 M70G 
                 SEQ ID NO: 203 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-53 
                 M70R 
                 SEQ ID NO: 204 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-54 
                 D72A 
                 SEQ ID NO: 205 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-55 
                 D72E 
                 SEQ ID NO: 206 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-56 
                 D72Q 
                 SEQ ID NO: 207 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-57 
                 D72R 
                 SEQ ID NO: 208 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-58 
                 D73A 
                 SEQ ID NO: 209 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-59 
                 Y36A + D72E 
                 SEQ ID NO: 192 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-60 
                 Y36A + L94R 
                 SEQ ID NO: 193 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-61 
                 E38A + D72E 
                 SEQ ID NO: 194 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-62 
                 E38A + L94K 
                 SEQ ID NO: 195 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-63 
                 E38A + L94R 
                 SEQ ID NO: 196 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-64 
                 E38R + D72R 
                 SEQ ID NO: 197 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-65 
                 D72E + L94K 
                 SEQ ID NO: 198 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                 R-kine-66 
                 D72E + L94R 
                 SEQ ID NO: 199 
                 SEQ ID NO: 101 
                 SEQ ID NO: 102 
               
               
                   
               
            
           
         
       
     
     For production of the immunocytokines, 6.0×10 6 /mL of ExpiCHO cells (ThermoFisher) with higher than 95% viability were prepared in 100 mL of cell culture media. 100 μg of the plasmid DNA encoding the immunocytokine was mixed with the ExpiFectamine™ CHO transfection reagent (ThermoFisher) and the mixture was added to the cell culture media. The cell culture was incubated in a platform shaker with the rotation rate at 150 rpm. The temperature was maintained at 37° C. while CO 2  level at 8%. 
     After ten days of incubation, the cells were pelleted by centrifuging at 4000 rpm, 25° C. for 10 minutes. Supernatant was collected for purification and gel electrophoresis. The supernatant was loaded on SDS-PAGE gel, following the instruction for NuPAGE™ 4-12% Bis-Tris Protein Gels (ThermoFisher). PageRuler™ Unstained Protein Ladder (ThermoFisher) was used alongside with the protein samples to determine the molecular weight of the protein. Fusion proteins were then purified by Protein A column (Cytiva) followed by SEC column (Cytiva). 
     7.4. Homogeneous Time-Resolved Fluorescence (HTRF) Phospho-STAT3 Assay of Immunocytokines (αPD-1IL21RαMutein/IL21) 
     The 66 immunocytokines were evaluated by measuring phosphorylation of STAT3 in HTRF-based high-throughput assay. Human cutaneous T lymphocyte cell lines (H9 (Cobioer), derivative of Hut78 cells) and H9 cells that stably expressing a programmed cell death protein 1 (PD-1(+) H9) were used in the pSTAT3 assay. Cells were grown in IMDM medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma Aldrich) for H9. 3 μg/mL puromycin (Invivogen) was additionally added for PD-1 positive H9 cells. Subculture of cells was conducted every 48 hours to avoid high density which could arrest the cell cycle. 
     Measuring pSTAT3 production was conducted to investigate the activation of cells by IL-21 binding with IL-21 receptors and common gamma chains. The high production of pSTAT3 was considered as a marker of strong reaction of treated materials. To conduct experiments, pSTAT3 ELISA kit (Perkin Elmer, MA) and Flex Station 3 (Molecular Devices, CA) were used following the manufacturer&#39;s user guide. 
     The detailed description is as follows. PD-1(−) H9 or PD-1(+) H9 cells were incubated with serum free media on overnight. After incubation, spin-down cells (with 125 g) were harvested with HBSS (Gibco) solution and seeded on white 96 well low volume plate (Cisbio) by 2.5×10 4  cells/well/8 μL. Compounds for evaluation were prepared with 3× concentration of final concentration and treated to cells for 30 minutes at 37° C. The lysis buffer was added to the wells for 30 minutes and then reagents for HTRF reaction were treated following the manufacturer&#39;s protocol. After 24 hours, the HTRF reaction was measured by Flex Station 3 equipment. 
     The non-linear analysis (4 parameters logistic regression) was conducted to calculate experiment parameters including EC 50 , Maximal response, and Hillslope. The Black and Leff operational model was adopted to estimate the compound&#39;s intrinsic efficacy. 
       FIGS.  4 ,  5 , and  6 A- 6 E  show that the moiety of an anti-PD-1 antibody of αPD-1IL21RαMutein/IL21 contributed to differences in the pharmacodynamics of αPD-1IL21RαMutein/IL21 on PD-1(+) or PD-1(−) H9 cells. 
     To be specific, the value of EC 50  of phosphorylation of STAT3 observed in αPD-1IL21RαMutein/IL21 treated PD-1(−) cells were higher than PD-1(+) cells, and the max efficacy was similar in both cell lines (Table 5). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Summary of EC 50   
               
            
           
           
               
               
               
            
               
                   
                 PD-1(−) H9 cells 
                 PD-1(+) H9 cells 
               
            
           
           
               
               
               
               
               
            
               
                 Mutation 
                 EC 50  (M) 
                 sem 
                 EC 50  (M) 
                 sem 
               
               
                   
               
               
                 Wildtype 
                 2.58E−07 
                 4.53E−08 
                 1.77E−07 
                 3.78E−08 
               
               
                 D72E 
                 1.57E−07 
                 2.03E−08 
                 3.29E−09 
                 5.80E−10 
               
               
                 A71R 
                 1.29E−07 
                 2.57E−08 
                 4.87E−09 
                 6.83E−10 
               
               
                 Y36G 
                 1.44E−07 
                 3.80E−08 
                 1.28E−08 
                 3.68E−09 
               
               
                 M70Q 
                 1.75E−07 
                 2.55E−08 
                 6.14E−09 
                 6.72E−10 
               
               
                 M70H 
                 1.98E−07 
                 3.57E−08 
                 6.93E−09 
                 8.64E−10 
               
               
                 M70W 
                 1.90E−07 
                 2.99E−08 
                 6.66E−09 
                 9.50E−10 
               
               
                 L94K 
                 2.53E−07 
                 5.90E−08 
                 1.03E−08 
                 1.88E−09 
               
               
                 E38R 
                 2.13E−07 
                 4.37E−08 
                 1.30E−09 
                 1.49E−10 
               
               
                 M70R 
                 1.63E−08 
                 3.00E−09 
                 3.21E−10 
                 4.36E−11 
               
               
                 M70D 
                 1.51E−07 
                 3.17E−08 
                 1.06E−09 
                 1.08E−10 
               
               
                 M70C 
                 2.96E−07 
                 6.20E−08 
                 1.15E−07 
                 2.63E−08 
               
               
                 M70G 
                 4.19E−07 
                 8.23E−08 
                 2.91E−09 
                 4.02E−10 
               
               
                 D72R 
                 1.20E−08 
                 2.60E−09 
                 5.04E−10 
                 6.72E−11 
               
               
                 D72Q 
                 1.65E−07 
                 3.35E−08 
                 9.50E−10 
                 1.17E−10 
               
               
                 D72A 
                 1.39E−07 
                 1.59E−08 
                 1.22E−09 
                 1.71E−10 
               
               
                 D73A 
                 6.45E−08 
                 9.86E−09 
                 6.13E−10 
                 8.81E−11 
               
               
                 D72E + E38A 
                 2.69E−08 
                 6.03E−09 
                 4.38E−10 
                 7.87E−11 
               
               
                   
               
            
           
         
       
     
     From the results of HTRF-based high-throughput screening, six variants of αPD-1IL21RαMutein/IL21, each containing a different mutein selected from E38R, M70D, M70H, M70Q, D72A, and L94K, were selected for further study ( FIG.  3   ). Among these six variants, M70Q and M70H muteins showed efficacy comparable to wild type IL-21, distinguishing from a fusion protein containing an attenuated IL-21 mentioned above. The attenuated IL-21 showed less than 80% efficacy compared to wild type IL-21 ( FIGS.  4  and  5   ). 
     These results demonstrate that IL-21Rα muteins of αPD-1IL21RαMutein/IL21 act as a capping molecule inhibiting IL-21 from binding to non-target cells, which is the reason for the low signal intensity in PD-1(−) cells. This shows that αPD-1IL21RαMutein/IL21 is an immunocytokine having high tissue specificity. 
     Besides, increasing specificity while maintaining efficacy of the drug substance by introducing a proper modification to a capping moiety to adjust specificity to its receptor is a unique advantage of our invention distinguishing from other drugs. αPD-1IL21RαMutein/IL21 shows characteristics of both full agonist and competitive antagonist. 
     7.5. SPR Full Kinetics Assay of Immunocytokines (αPD-1IL21Rα Mutein/IL21) against PD-1 
     Interaction between immunocytokine and human PD-1 (hPD-1) was determined by Surface Plasmon Resonance (SPR, Biacore 8K) analysis. Immunocytokines and hPD-1&#39;s affinity was tested by CMS sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CM5 sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 25 μg/mL of anti-human Fc IgG in 10 mM NaAc (pH 4.5) to the channel 1-8 for 420 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s. 
     Immunocytokines diluted in running buffer (1× HBS-EP+) were captured on to Fc2 via anti-human Fc IgG at flow rate of 10 μL/min for 40 s. Multiple cycle kinetics was used to perform the assay. The analyte hPD-1 at 7 different concentrations (0, 2.5, 5, 10, 20, 40, and 80 nM) and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 180 s, followed by 900 s dissociation. 10 mM glycine pH 1.5 was injected as regeneration buffer following every dissociation phase. 
     The sensorgrams from the reference channel Fc1 and the buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model. Molecular weight of 17 kDa were used to calculate the molar concentration of hPD-1. 
     The SPR analysis demonstrated that the fusion of IL21RαWT or IL21RαMutein and IL-21 to the anti-PD-1 antibody did not affect the affinity of the anti-PD-1 antibody to PD-1 ( FIG.  7 A- 7 Q , Table 6). 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 No. 
                 IL-21R Mutation Site 
                 K D  (M) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 WT 
                 8.41E−09 
               
               
                 2 
                 Y36C 
                 8.36E−09 
               
               
                 3 
                 Y36E 
                 8.32E−09 
               
               
                 4 
                 Y36G 
                 8.51E−09 
               
               
                 5 
                 Y36H 
                 9.02E−09 
               
               
                 6 
                 Y36I 
                 8.74E−09 
               
               
                 7 
                 Y36K 
                 8.23E−09 
               
               
                 8 
                 Y36M 
                 8.32E−09 
               
               
                 9 
                 Y36N 
                 8.20E−09 
               
               
                 10 
                 Y36P 
                 8.44E−09 
               
               
                 11 
                 Y36Q 
                 8.49E−09 
               
               
                 12 
                 Y36R 
                 8.45E−09 
               
               
                 13 
                 Y36S 
                 8.39E−09 
               
               
                 14 
                 Y36T 
                 8.52E−09 
               
               
                 15 
                 Y36V 
                 8.63E−09 
               
               
                 16 
                 E38C 
                 9.06E−09 
               
               
                 17 
                 E38R 
                 6.61E−09 
               
               
                 18 
                 E38Y 
                 8.73E−09 
               
               
                 19 
                 L39C 
                 8.61E−09 
               
               
                 20 
                 L39E 
                 8.45E−09 
               
               
                 21 
                 L39H 
                 8.24E−09 
               
               
                 22 
                 L39K 
                 8.43E−09 
               
               
                 23 
                 L39R 
                 8.62E−09 
               
               
                 24 
                 L39W 
                 8.78E−09 
               
               
                 25 
                 L39Y 
                 9.74E−09 
               
               
                 26 
                 M70F 
                 9.01E−09 
               
               
                 27 
                 M70H 
                 8.85E−09 
               
               
                 28 
                 M70N 
                 8.78E−09 
               
               
                 29 
                 M70Q 
                 8.85E−09 
               
               
                 30 
                 M70S 
                 8.84E−09 
               
               
                 31 
                 M70T 
                 8.49E−09 
               
               
                 32 
                 M70V 
                 8.53E−09 
               
               
                 33 
                 M70W 
                 8.86E−09 
               
               
                 34 
                 M70Y 
                 8.81E−09 
               
               
                 35 
                 A71E 
                 8.14E−09 
               
               
                 36 
                 A71F 
                 8.70E−09 
               
               
                 37 
                 A71I 
                 8.59E−09 
               
               
                 38 
                 A71L 
                 8.87E−09 
               
               
                 39 
                 A71Q 
                 8.51E−09 
               
               
                 40 
                 A71R 
                 8.94E−09 
               
               
                 41 
                 A71W 
                 9.12E−09 
               
               
                 42 
                 A71Y 
                 8.94E−09 
               
               
                 43 
                 I74R 
                 8.90E−09 
               
               
                 44 
                 I74W 
                 8.94E−09 
               
               
                 45 
                 L94F 
                 8.39E−09 
               
               
                 46 
                 L94K 
                 8.90E−09 
               
               
                 47 
                 L94Q 
                 8.90E−09 
               
               
                 48 
                 L94R 
                 7.82E−09 
               
               
                 49 
                 L94Y 
                 9.19E−09 
               
               
                 50 
                 M70C 
                 7.91E−09 
               
               
                 51 
                 M70D 
                 7.25E−09 
               
               
                 52 
                 M70G 
                 8.42E−09 
               
               
                 53 
                 M70R 
                 6.83E−09 
               
               
                 54 
                 D72A 
                 6.88E−09 
               
               
                 55 
                 D72E 
                 6.53E−09 
               
               
                 56 
                 D72Q 
                 8.64E−09 
               
               
                 57 
                 D72R 
                 8.03E−09 
               
               
                 58 
                 D73A 
                 7.47E−09 
               
               
                 59 
                 Y36A + L94R 
                 7.26E−09 
               
               
                 60 
                 E38A + L94K 
                 7.70E−09 
               
               
                 61 
                 E38A + L94R 
                 8.16E−09 
               
               
                 62 
                 D72E + Y36A 
                 7.39E−09 
               
               
                 63 
                 D72E + E38A 
                 7.19E−09 
               
               
                 64 
                 D72E + L94K 
                 7.58E−09 
               
               
                 65 
                 D72E + L94R 
                 8.01E−09 
               
               
                 66 
                 D72R + E38R 
                 7.02E−09 
               
               
                   
               
            
           
         
       
     
     7.6. Binding Affinity of Immunocytokines (αPD-1IL21RαMutein/IL21) to FcRn 
     The binding affinity of antibody-based protein drugs to FcRn is known to be highly associated with its half-life in vivo. The binding affinity of immunocytokines (αPD-1IL21RαMutein/IL21) to FcRn was measured using Bio-Layer Interferometry (BLI) system. As a control, the binding affinity of anti-PD-1 antibody which is not conjugated to IL-21 or IL-21 RαMutein was also measured. 
     For the assay, FAB2G biosensor (Sartorius) was hydrated with a running buffer for 10 minutes in the 96 well plate (Corning). The ligands (anti-PD-1 antibody or Immunocytokine) were diluted with the running buffer to make a final concentration of 0.5 μg/ml for anti-PD-1 antibody and 2 μg/ml for immunocytokine. FAB2G biosensor was loaded with either anti-PD-1 antibody or Immunocytokine at 1.5nm level. After loading either anti-PD-1 antibody or Immunocytokine, the baseline was set by incubating the loaded sensor tip in the running buffer for 300 sec. Ligand loaded sensor tips were incubated in wells containing a 2-fold serial dilution of soluble, FcRn/B2M complex receptors. Association and dissociation were measured for 60 seconds or until a steady state was reached. The measurement data are provided in  FIG.  8 A . 
     The binding affinities of the anti-PD-1 antibody or Immunocytokine to FcRn were measured using Octet RED96e (ForteBio) instruments. Optimized Octet sample buffer (100 mM Sodium Phosphate, 300 mM NaCl, 0.05% Tween20) was used for sample dilution and all binding baseline, association, and dissociation steps at either pH of 6.0 or pH of 7.4. A buffer only blank curve was subtracted to correct any drift. The data were fit to a 1:1 binding model using ForteBio data analysis software 11.1 to determine the K on , K off , and K D , which are provided in  FIG.  8 B . 
     The data show that the binding affinity of the immunocytokine to FcRn is not significantly different from the binding affinity of the anti-PD-1 antibody. This result suggests that the pharmacokinetic profile of the instant immunocytokine will benefit from FcRn binding ability, thus having a half-life sufficient to provide therapeutic effects. 
     7.7. In Vitro Tumor Killing Assay 
     To confirm the anti-tumor effects of the present immunocytokine (αPD-1IL21RαMutein/IL21), an increase in IFNγ expression level and a change in cytotoxicity of the CD8+ T cells that are treated with the present immunocytokine were tested. When the CD8+ T cells are co-cultured with autologous monocyte-derived DCs (moDCs) presenting specific antigens on their surfaces through MHC-peptide complexes, the tumor antigen educated CD8+ T cells (e.g., CTLs) can recognize and attack tumor cells expressing those antigens. The efficacy of the immunocytokines was confirmed by measuring fluorescent materials leaked from the tumor cells due to the death of tumor cells. 
     Specifically, human PBMCs were purchased from StemExpress (USA). Monocytes were isolated using Pan Monocyte Isolation Kit (Miltenyi Biotec) and were cultured for 7 days with 35 ng/mL recombinant human IL-4 (R&amp;D Systems) and 50 ng/mL GM-CSF (R&amp;D Systems) in RPMI1640 medium(Gibco) to differentiate the monocyte to dendritic cells (DCs). The premature monocyte-derived DCs were further matured for 3 days using 10 ng/mL recombinant human IL-6 (R&amp;D Systems), 15 ng/mL IL-1β (R&amp;D Systems), 40 ng/mL TGFα (R&amp;D Systems), and 1 μg/mL PGE2 (PeproTech). During maturation, antigen peptides were loaded on the monocyte-derived DCs (moDCs). Autologous donor&#39;s CD8+ T cells were isolated using CD8 +  T Cell Isolation Kit (Miltenyi Biotec) and were co-cultured with the matured moDCs for 10 days at a 10:1 cell number ratio. Culture medium supplemented with recombinant human IL-15 (R&amp;D Systems) and recombinant human IL-7 (R&amp;D Systems) were added every 2 or 3 days to sustain CTLs. 
     CTLs were then expanded using an anti-CD3ε antibody (R&amp;D Systems), anti-CD28 antibody (R&amp;D Systems), and recombinant human IL-2 (R&amp;D Systems) for 5 days. During the expansion of CTLs (effector cell), the present immunocytokines (αPD-1IL21RαMutein/IL21 or αPD-1IL21RαWT/IL21) or controls (e.g., anti-PD-1 antibody) were treated at 500nM concentration. 
     7.7.1. Release of IFN-γ 
     IFNγ levels in the culture supernatants were measured by ELISA using Human IFN-gamma DuoSet ELISA kit (R&amp;D Systems). The results are provided in  FIG.  10   , confirming increased IFNγ release from CTL in response to immunocytokines (four variants of αPD-1IL21RαMutein/IL21, each containing a different mutein selected from M70D, M70Q, L94K, and E39R). 
     7.7.2. Cytotoxicity 
     To confirm tumor killing efficacy, Calcein AM(Invitrogen)-stained target cells (MeWo cell line or CMV pp65 gene transduced A375 cell line (A375_CMV)) were plated the day before co-culture with the expanded CTLs (effector cells). The effector cells were collected and loaded to the medium with target cells and cultured for 36 hours. The release of Calcein AM from the dead tumor cells were measured by detecting fluorescent signals at Ex 485 nm and Em 530 nm using FlexStation3 equipment. 
       FIGS.  11 A and  11 B  provide data from MeWo cell line and A375_CMV cell line, respectively. The data show that CTLs treated with αPD-1IL21RαMutein/IL21 showed better tumor-killing activity than the controls. This can be due to enhancement of effector function of CTLs by αPD-1IL21RαMutein/IL21. These suggest that immunocytokines provided here, αPD-1IL21RαMutein/IL21, can enhance anti-tumor activity when applied to cancer patients. 
     7.8. Immunocytokines against CTLA-4, TIGIT, LAG-3 (αCTLA-4L21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) 
     Two immunocytokines against each of three different targets, CTLA-4, TIGIT, and LAG-3 (αCTLA-4L21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) were generated by methods described above related to αPD-1L21RαMutein/IL21. The immunocytokines includes (i) a first chain comprising a heavy chain, G4S linker and IL-21RαMutein; (ii) a second chain comprising a heavy chain, G4S linker and a human IL-21; and (iii) two light chains, as specified in Table 7. The immunocytokines were successfully generated from the CHO cell lines, and the HTRF assay confirmed their functional activity of phosphorylation of STAT3 as described in 5.9. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                 Sequence of first 
                 Sequence of second 
                   
               
               
                   
                 IL-21Rα 
                 chain (Heavy 
                 chain (Heavy 
               
               
                   
                 Mutation 
                 Chain-G4S Linker- 
                 Chain-G4S Linker- 
                 Sequence of two 
               
               
                   
                 Site 
                 IL-21RαMutein) 
                 human IL-21) 
                 light chains 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Ipilimumab 
                 WT 
                 SEQ ID NO: 230 
                 SEQ ID NO: 229 
                 SEQ ID NO: 152 
               
               
                   
                 M70D 
                 SEQ ID NO: 231 
                 SEQ ID NO: 229 
                 SEQ ID NO: 152 
               
               
                   
                 D72A 
                 SEQ ID NO: 232 
                 SEQ ID NO: 229 
                 SEQ ID NO: 152 
               
               
                 Tiragolumab 
                 WT 
                 SEQ ID NO: 234 
                 SEQ ID NO: 233 
                 SEQ ID NO: 226 
               
               
                   
                 M70D 
                 SEQ ID NO: 235 
                 SEQ ID NO: 233 
                 SEQ ID NO: 226 
               
               
                   
                 D72A 
                 SEQ ID NO: 236 
                 SEQ ID NO: 233 
                 SEQ ID NO: 226 
               
               
                 Relatlimab 
                 WT 
                 SEQ ID NO: 238 
                 SEQ ID NO: 237 
                 SEQ ID NO: 228 
               
               
                   
                 M70D 
                 SEQ ID NO: 239 
                 SEQ ID NO: 237 
                 SEQ ID NO: 228 
               
               
                   
                 D72A 
                 SEQ ID NO: 240 
                 SEQ ID NO: 237 
                 SEQ ID NO: 228 
               
               
                   
               
            
           
         
       
     
     7.9. Homogeneous Time-Resolved Fluorescence (HTRF) Phosphor-STAT3 Assay of Immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; and αLAG-3IL21RαMutein/IL21) 
     The immunocytokines against CTLA-4, TIGIT or LAG-3 were evaluated by measuring phosphorylation of STAT3 in HTRF-based high-throughput assay. Human cutaneous T lymphocyte cell lines (H9 (Cobioer), derivative of Hut78 cells) were grown in IMDM medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma Aldrich) for H9. 3 μg/mL puromycin (Invivogen) was additionally added to the H9 cells. Subculture of cells was conducted every 48 hours to avoid high density which could arrest the cell cycle. 
     H9 cells were incubated with serum free media on overnight. After incubation, spin-down cells (with 125 g) were harvested with HBSS (Gibco) solution and seeded on white 96 well low volume plate (Cisbio) by 2.5×10 4  cells/well/8 μL. Compounds for evaluation were prepared at 3× of the final concentration and applied to cells for 30 minutes at 37° C. The lysis buffer was added to the wells for 30 minutes and then reagents for HTRF reaction were treated following the manufacturer&#39;s protocol. After 24 hours, the HTRF reaction was measured by Flex Station 3 equipment. 
       FIG.  12    and Table 8 provide data demonstrating that rhIL21, ABP-IL21RαWT/IL21, and ABP-IL21RαMutein/IL21 activated HTRF reaction. Among them, ABP-IL21RαWT/IL21 and ABP-IL21RαMutein/IL21 had significant lower activity than rhIL21, because of the masking effects of IL21RαWT or IL21RαMutein against IL21. As expected given that IL21RαWT has a higher affinity to IL21 compared to IL21RαMutein, the masking effects of IL21RαWT were greater than IL21RαMutein. 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Summary of EC 50   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 αCTLA-4 
                 αCTLA-4 
                 αCTLA-4 
               
               
                   
                   
                 anti-CTLA- 
                 IL21Rα 
                 IL21RαMutein 
                 IL21RαMutein 
               
               
                   
                 rhIL21 
                 4 antibody 
                 WT/IL21 
                 (M70D)/IL21 
                 (D72A)/IL21 
               
               
                   
               
               
                 EC50(M) 
                 7.47E−10 
                 N/A 
                 6.524E−07 
                 3.399E−07 
                 5.143E−07 
               
               
                 EC50 ratio 
                 1.0 
                 N/A 
                 873.4 
                 455.0 
                 688.5 
               
               
                   
               
               
                   
                   
                 anti- 
                 αTIGIT 
                 αTIGIT 
                 αTIGIT 
               
               
                   
                   
                 TIGIT 
                 IL21Rα 
                 IL21RαMutein 
                 IL21RαMutein 
               
               
                   
                   
                 antibody 
                 WT/IL21 
                 (M70D)/IL21 
                 (D72A)/IL21 
               
               
                   
               
               
                 EC50(M) 
                   
                 N/A 
                 6.096E−07 
                 2.017E−07 
                 1.67E−07 
               
               
                 EC50 ratio 
                   
                 N/A 
                 816.1 
                 270.0 
                 223.6 
               
               
                   
               
               
                   
                   
                   
                 αLAG-3 
                 αLAG-3 
                 αLAG-3 
               
               
                   
                   
                 anti-LAG- 
                 IL21Rα 
                 IL21RαMutein 
                 IL21RαMutein 
               
               
                   
                   
                 3 antibody 
                 WT/IL21 
                 (M70D)/IL21 
                 (D72A)/IL21 
               
               
                   
               
               
                 EC50(M) 
                   
                 N/A 
                 2.73E−06 
                 1.42E−07 
                 4.538E−07 
               
               
                 EC50 ratio 
                   
                 N/A 
                 3654.6  
                 190.1 
                 607.5 
               
               
                   
               
            
           
         
       
     
     7.10. SPR Full Kinetics Assay of Immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) 
     Binding between the immunocytokines and their respective human target proteins (hCTLA-4, hTIGIT, or hLAG-3) was tested by Surface Plasmon Resonance (SPR) analysis. Affinities of the immunocytokines to their human ligands were tested by CM5 sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CM5 sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 25 μg/mL of anti-human Fc IgG in 10 mM NaAc (pH 4.5) to the channel 1-8 for 420 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s. 
     Immunocytokines diluted in running buffer (1× HBS-EP+) were captured on to Fc2 via anti-human Fc IgG at flow rate of 10 μL/min for 40 s. Multiple cycle kinetics was used to perform the assay. 6 concentrations (1.56, 3.13, 6.25, 12.5, 25, and 50 nM) of analyte hCTLA-4 (Acro Biosystems) or 6 concentrations (0.78, 1.56, 3.13, 6.25, 12.5, and 25 nM) of analyte hTIGIT (R&amp;D systems) or 6 concentrations (0.31, 0.63, 1.25, 2.5, 5, and 10nM) of analyte hLAG-3 (Acro Biosystems) and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 180 s, followed by 900 s dissociation. 10 mM glycine pH 1.5 was injected as a regeneration buffer following every dissociation phase. 
     The sensorgrams for reference channel Fc1 and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model or heterogeneous ligand model. 
     The SPR analysis demonstrated that the fusion of IL21RαWT or IL21RαMutein and IL-21 to the anti-CTLA-4, anti-TIGIT or anti-LAG-3 antibody did not affect the affinity of the anti-CTLA-4, anti-TIGIT or anti-LAG-3 antibody to its respective target (Table 9;  FIGS.  9 A,  9 B and  9 C ). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Affinity of immunocytokines (αCTLA-4IL21RαMutein/IL21; 
               
               
                 αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) 
               
               
                 against targets (CTLA-4; TIGIT; or LAG-3) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Control 
                   
                   
                 IL-21R 
                   
               
               
                 No. 
                 Antibody 
                 K D  (M) 
                 No. 
                 Mutation Site 
                 K D  (M) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 Ipilimumab 
                 2.09E−08 
                 Against 
               
               
                   
                   
                   
                 hCTLA-4 
               
            
           
           
               
               
               
               
               
               
            
               
                 2 
                 Tiragolumab 
                 5.95E−11 
                 1 
                 WT 
                 1.80E−08 
               
               
                 3 
                 Relatlimab 
                 2.39E−10 
                 2 
                 M70D 
                 1.81E−08 
               
            
           
           
               
               
               
               
            
               
                 N/A 
                 3 
                 D72A 
                 1.71E−08 
               
            
           
           
               
               
            
               
                   
                 Against 
               
               
                   
                 hTIGIT 
               
            
           
           
               
               
               
               
            
               
                   
                 4 
                 WT 
                 5.17E−11 
               
               
                   
                 5 
                 M70D 
                 5.37E−11 
               
               
                   
                 6 
                 D72A 
                 5.60E−11 
               
            
           
           
               
               
            
               
                   
                 Against 
               
               
                   
                 hLAG-3 
               
            
           
           
               
               
               
               
            
               
                   
                 7 
                 WT 
                 2.64E−10 
               
               
                   
                 8 
                 M70D 
                 2.49E−10 
               
               
                   
                 9 
                 D72A 
                 2.55E−10 
               
               
                   
                   
               
            
           
         
       
     
     8. EQUIVALENTS AND INCORPORATION BY REFERENCE 
     While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. 
     All references, issued patents and patent applications cited within the body of the instant specification, are hereby incorporated by reference in their entirety, for all purposes. 
     9. SEQUENCE LISTING 
       
     
       
         
           
               
            
               
                   
               
               
                 Summary of Sequence Listing 
               
            
           
           
               
               
            
               
                 SEQ 
                   
               
               
                 ID NO 
                 Sequences 
               
               
                   
               
               
                  1 
                 Nivolumab heavy chain 
               
               
                  2 
                 Nivolumab light chain 
               
               
                  3 
                 pembrolizumab heavy chain 
               
               
                  4 
                 pembrolizumab light chain 
               
               
                  5 
                 cemiplimab heavy chain 
               
               
                  6 
                 cemiplimab light chain 
               
               
                  7 
                 atezolizumab heavy chain 
               
               
                  8 
                 atezolizumab light chain 
               
               
                  9 
                 dostarlimab heavy chain 
               
               
                  10 
                 dostarlimab light chain 
               
               
                  11 
                 durvalumab heavy chain 
               
               
                  12 
                 durvalumab light chain 
               
               
                  13 
                 avelumab heavy chain 
               
               
                  14 
                 avelumab light chain 
               
               
                  15 
                 Wild type IL-21Rα (ectodomain; extracellular domain) 
               
               
                  16 
                 Human IgG1 Fc (100 Pro-330 Lys) 
               
               
                  17 
                 G4S linker 
               
               
                 18-99 
                 IL-21RαMutein 
               
               
                 100 
                 Human IL-21 
               
               
                 101 
                 Second Chain (Heavy chain of anti-PD-1 antibody + linker + 
               
               
                   
                 human IL-21) with knob mutation 
               
               
                 102 
                 Anti-PD-1 antibody, Light chain 
               
               
                 103 
                 Anti-PD-1 antibody, Heavy chain with hole mutation 
               
               
                 104-150 
                 αPD-1 + linker + IL21RαMutein 
               
               
                 151 
                 (Ipilimumab heavy chain) 
               
               
                 152 
                 (Ipilimumab light chain) 
               
               
                 153 
                 (tremelimumab heavy chain) 
               
               
                 154 
                 (tremelimumab light chain) 
               
               
                 155-169 
                 IL21RαMutein 
               
               
                 170-184 
                 Fc-Linker-IL21RαMutein 
               
               
                 185 
                 IgG1 Fc moiety (WT) 
               
               
                 186 
                 IgG2 Fc moiety (WT) 
               
               
                 187 
                 IgG3 Fc moiety (WT) 
               
               
                 188 
                 IgG4 Fc moiety (WT) 
               
               
                 189 
                 IGHG1 (Immunoglobulin heavy constant gamma 1) with 
               
               
                   
                 ‘LALA’(L234A/L235A) mutation 
               
               
                 190 
                 IGHG4 (Immunoglobulin heavy constant gamma 4) with 
               
               
                   
                 ‘SPLE’(S228P/L235E) mutation 
               
               
                 191 
                 αPD-1-linker- IL21RαWT 
               
               
                 192-209 
                 αPD-1 + linker + IL21RαMutein 
               
               
                 210 
                 Polynucleotide encoding IgG1-1IL21Rα (wild type) 
               
               
                 211 
                 Polynucleotide encoding αPD-1IL21Rα (wild type) 
               
               
                 212-217 
                 Flexible linkers 
               
               
                 218-224 
                 Rigid linkers 
               
               
                 225 
                 Tiragolumab Heavy chain 
               
               
                 226 
                 Tiragolumab light chain 
               
               
                 227 
                 Relatlimab Heavy chain 
               
               
                 228 
                 Relatlimab Light chain 
               
               
                 229 
                 Second Chain (Heavy chain of anti-CTLA-4 antibody + 
               
               
                   
                 linker + human IL-21) with knob mutation 
               
               
                 230 
                 αCTLA-4 + linker + IL21RαWT 
               
               
                 231-232 
                 αCTLA-4 + linker + IL21RαMutein 
               
               
                 233 
                 Second Chain (Heavy chain of anti-TIGIT antibody + 
               
               
                   
                 linker + human IL-21) with knob mutation 
               
               
                 234 
                 αTIGIT + linker + IL21RαWT 
               
               
                 235-236 
                 αTIGIT + linker + IL21RαMutein 
               
               
                 237 
                 Second Chain (Heavy chain of anti-LAG-3 antibody + 
               
               
                   
                 linker + human IL-21) with knob mutation 
               
               
                 238 
                 αLAG-3 + linker + IL21RαWT 
               
               
                 239-240 
                 αLAG-3 + linker + IL21RαMutein 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                   
               
               
                 SEQ ID NO 
                 Sequence 
               
               
                   
               
             
            
               
                 1 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 (Nivolumab 
                 LEWVAVIWYDGSKRYY 
               
               
                 heavy chain) 
                 ADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWG 
               
               
                   
                 QGTLVTVSSASTKGPS 
               
               
                   
                 VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF 
               
               
                   
                 PAVLQSSGLYSLSS 
               
               
                   
                 VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE 
               
               
                   
                 FLGGPSVFLFPPKP 
               
               
                   
                 KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                 REEQFNSTYRVVSVLT 
               
               
                   
                 VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP 
               
               
                   
                 PSQEEMTKNQVSLTC 
               
               
                   
                 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD 
               
               
                   
                 KSRWQEGNVFSCSV 
               
               
                   
                 MHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 2 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                 (Nivolumab light 
                 IYDASNRATGIPA 
               
               
                 chain) 
                 RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 
               
               
                   
                 RTVAAPSVFIFPP 
               
               
                   
                 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT 
               
               
                   
                 EQDSKDSTYSLSSTLT 
               
               
                   
                 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 3 
                 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQ 
               
               
                 (pembrolizumab 
                 GLEWMGGINPSNGGTNF 
               
               
                 heavy chain) 
                 NEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDM 
               
               
                   
                 GFDYWGQGTTVTVSS 
               
               
                   
                 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL 
               
               
                   
                 TSGVHTFPAVLQSS 
               
               
                   
                 GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP 
               
               
                   
                 CPPCPAPEFLGGPSV 
               
               
                   
                 FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH 
               
               
                   
                 NAKTKPREEQFNSTY 
               
               
                   
                 RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE 
               
               
                   
                 PQVYTLPPSQEEMTK 
               
               
                   
                 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL 
               
               
                   
                 YSRLTVDKSRWQEG 
               
               
                   
                 NVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 4 
                 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQ 
               
               
                 (pembrolizumab 
                 APRLLIYLASYLES 
               
               
                 light chain) 
                 GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTK 
               
               
                   
                 VEIKRTVAAPSVF 
               
               
                   
                 IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE 
               
               
                   
                 SVTEQDSKDSTYSLS 
               
               
                   
                 STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 5 
                 EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA 
               
               
                 (cemiplimab 
                 PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY 
               
               
                 heavy chain) 
                 LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST 
               
               
                   
                 KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS 
               
               
                   
                 GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC 
               
               
                   
                 NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF 
               
               
                   
                 PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE 
               
               
                   
                 VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV 
               
               
                   
                 SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV 
               
               
                   
                 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 
               
               
                   
                 FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL 
               
               
                   
                 SLGK 
               
               
                   
               
               
                 6 
                 DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP 
               
               
                 (cemiplimab light 
                 GKAPNLLIYA ASSLHGGVPS 
               
               
                 chain) 
                 RFSGSGSGTD FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP 
               
               
                   
                 GTVVDFRRTV AAPSVFIFPP 
               
               
                   
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
                 ESVTEQDSKD STYSLSSTLT 
               
               
                   
                 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 
               
               
                   
               
               
                 7 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGL 
               
               
                 (atezolizumab 
                 EWVAWISPYGGSTYY 
               
               
                 heavy chain) 
                 ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGG 
               
               
                   
                 FDYWGQGTLVTVSSAS 
               
               
                   
                 TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 
               
               
                   
                 GVHTFPAVLQSSGL 
               
               
                   
                 YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH 
               
               
                   
                 TCPPCPAPELLGGPS 
               
               
                   
                 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV 
               
               
                   
                 HNAKTKPREEQYAST 
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR 
               
               
                   
                 EPQVYTLPPSREEMT 
               
               
                   
                 KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF 
               
               
                   
                 LYSKLTVDKSRWQQ 
               
               
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 8 
                 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK 
               
               
                 (atezolizumab 
                 LLIYSASFLYSGVPS 
               
               
                 light chain) 
                 RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK 
               
               
                   
                 RTVAAPSVFIFPP 
               
               
                   
                 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT 
               
               
                   
                 EQDSKDSTYSLSSTLT 
               
               
                   
                 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 9 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGL 
               
               
                 (dostarlimab 
                 EWVSTISGGGSYTYY 
               
               
                 heavy chain) 
                 QDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMD 
               
               
                   
                 YWGQGTTVTVSSASTK 
               
               
                   
                 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 
               
               
                   
                 HTFPAVLQSSGLYS 
               
               
                   
                 LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP 
               
               
                   
                 APEFLGGPSVFLFP 
               
               
                   
                 PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK 
               
               
                   
                 TKPREEQFNSTYRVVS 
               
               
                   
                 VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY 
               
               
                   
                 TLPPSQEEMTKNQVS 
               
               
                   
                 LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL 
               
               
                   
                 TVDKSRWQEGNVFS 
               
               
                   
                 CSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 10 
                 DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKL 
               
               
                 (dostarlimab light 
                 LIYWASTLHTGVPS 
               
               
                 chain) 
                 RFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIK 
               
               
                   
                 RTVAAPSVFIFPP 
               
               
                   
                 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT 
               
               
                   
                 EQDSKDSTYSLSSTLT 
               
               
                   
                 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 11 
                 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKG 
               
               
                 (durvalumab 
                 LEWVANIKQDGSEKYY 
               
               
                 heavy chain) 
                 VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFG 
               
               
                   
                 ELAFDYWGQGTLVTVS 
               
               
                   
                 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 
               
               
                   
                 LTSGVHTFPAVLQS 
               
               
                   
                 SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK 
               
               
                   
                 THTCPPCPAPEFEG 
               
               
                   
                 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG 
               
               
                   
                 VEVHNAKTKPREEQY 
               
               
                   
                 NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG 
               
               
                   
                 QPREPQVYTLPP SRE 
               
               
                   
                 EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD 
               
               
                   
                 GSFFLYSKLTVDKSR 
               
               
                   
                 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 12 
                 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPR 
               
               
                 (durvalumab light 
                 LLIYDASSRATGIP 
               
               
                 chain) 
                 DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVE 
               
               
                   
                 IKRTVAAPSVFIFP 
               
               
                   
                 PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV 
               
               
                   
                 TEQDSKDSTYSLSSTL 
               
               
                   
                 TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 13 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGL 
               
               
                 (avelumab heavy 
                 EWVSSIYPSGGITFY 
               
               
                 chain) 
                 ADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVT 
               
               
                   
                 TVDYWGQGTLVTVSS 
               
               
                   
                 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL 
               
               
                   
                 TSGVHTFPAVLQSS 
               
               
                   
                 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT 
               
               
                   
                 HTCPPCPAPELLGG 
               
               
                   
                 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV 
               
               
                   
                 EVHNAKTKPREEQYN 
               
               
                   
                 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ 
               
               
                   
                 PREPQVYTLPPSRDE 
               
               
                   
                 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
                 FFLYSKLTVDKSRW 
               
               
                   
                 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 14 
                 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP 
               
               
                 (avelumab light 
                 KLMIYDVSNRPSGV 
               
               
                 chain) 
                 SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTK 
               
               
                   
                 VTVLGQPKANPTVT 
               
               
                   
                 LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETT 
               
               
                   
                 KPSKQSNNKYAASS 
               
               
                   
                 YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
               
               
                 15 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 IL-21RαWT 
                 CSLHRSAHNATHATY 
               
               
                   
                 TCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFN 
               
               
                   
                 VTVTFSGQYNISWR 
               
               
                   
                 SDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVS 
               
               
                   
                 LLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEE 
               
               
                   
                 LKE 
               
               
                   
               
               
                 16 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 Human IgG1 Fc 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (100 Pro-330 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 Lys) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGK 
               
               
                   
               
               
                 17 
                 GGGGSGGGGSGGGGS 
               
               
                 G4S linker 
                   
               
               
                   
               
               
                 18 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQD K YEELKDEATS 
               
               
                 Q35K 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 19 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQD R YEELKDEATS 
               
               
                 Q35R 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 20 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQD Y YEELKDEATS 
               
               
                 Q35Y 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 21 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ C EELKDEATS 
               
               
                 Y36C 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 22 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ E EELKDEATS 
               
               
                 Y36E 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 23 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ G EELKDEATS 
               
               
                 Y36G 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 24 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ H EELKDEATS 
               
               
                 Y36H 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 25 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ I EELKDEATSC 
               
               
                 Y36I 
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                 (IL-21Rα mutein) 
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 26 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ K EELKDEATS 
               
               
                 Y36K 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 27 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ M EELKDEATS 
               
               
                 Y36M 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 28 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ N EELKDEATS 
               
               
                 Y36N 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 29 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ P EELKDEATSC 
               
               
                 Y36P 
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                 (IL-21Rα mutein) 
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 30 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ Q EELKDEATS 
               
               
                 Y36Q 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 31 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ R EELKDEATS 
               
               
                 Y36R 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 32 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ S EELKDEATSC 
               
               
                 Y36S 
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                 (IL-21Rα mutein) 
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 33 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ T EELKDEATS 
               
               
                 Y36T 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 34 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ V EELKDEATS 
               
               
                 Y36V 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 35 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE C LKDEATS 
               
               
                 E38C 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 36 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE K LKDEATS 
               
               
                 E38K 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 37 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYERLKDEATS 
               
               
                 E38R 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 38 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE Y LKDEATS 
               
               
                 E38Y 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 39 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE C KDEATS 
               
               
                 L39C 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 40 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE E KDEATS 
               
               
                 L39E 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 41 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE F KDEATS 
               
               
                 L39F 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 42 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE H KDEATS 
               
               
                 L39H 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 43 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE K KDEATS 
               
               
                 L39K 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 44 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE R KDEATS 
               
               
                 L39R 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 45 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE W KDEATS 
               
               
                 L39W 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 46 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE Y KDEATS 
               
               
                 L39Y 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 47 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70C 
                 CSLHRSAHNATHATYTCHMDVFHF C ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 48 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70D 
                 CSLHRSAHNATHATYTCHMDVFHF D ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 49 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70F 
                 CSLHRSAHNATHATYTCHMDVFHF F ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 50 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70G 
                 CSLHRSAHNATHATYTCHMDVFHF G ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 51 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70H 
                 CSLHRSAHNATHATYTCHMDVFHF H ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 52 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70K 
                 CSLHRSAHNATHATYTCHMDVFHF K ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 53 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70L 
                 CSLHRSAHNATHATYTCHMDVFHF L ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 54 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70N 
                 CSLHRSAHNATHATYTCHMDVFHF N ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 55 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70Q 
                 CSLHRSAHNATHATYTCHMDVFHF Q ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 56 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70R 
                 CSLHRSAHNATHATYTCHMDVFHF R ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 57 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70S 
                 CSLHRSAHNATHATYTCHMDVFHF S ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 58 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70T 
                 CSLHRSAHNATHATYTCHMDVFHF T ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 59 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70V 
                 CSLHRSAHNATHATYTCHMDVFHF V ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 60 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70W 
                 CSLHRSAHNATHATYTCHMDVFHF W ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 61 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M70Y 
                 CSLHRSAHNATHATYTCHMDVFHF Y ADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 62 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71E 
                 CSLHRSAHNATHATYTCHMDVFHFM E DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 63 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71F 
                 CSLHRSAHNATHATYTCHMDVFHFM F DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 64 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71I 
                 CSLHRSAHNATHATYTCHMDVFHFM I DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 65 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71L 
                 CSLHRSAHNATHATYTCHMDVFHFM L DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 66 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71Q 
                 CSLHRSAHNATHATYTCHMDVFHFM Q DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 67 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71R 
                 CSLHRSAHNATHATYTCHMDVFHFM R DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 68 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71W 
                 CSLHRSAHNATHATYTCHMDVFHFM W DDIFSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 69 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 A71Y 
                 CSLHRSAHNATHATYTCHMDVFHFM Y DDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 70 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72A 
                 CSLHRSAHNATHATYTCHMDVFHFMA A DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 71 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72C 
                 CSLHRSAHNATHATYTCHMDVFHFMA C DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 72 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72E 
                 CSLHRSAHNATHATYTCHMDVFHFMA E DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 73 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72F 
                 CSLHRSAHNATHATYTCHMDVFHFMA F DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 74 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72G 
                 CSLHRSAHNATHATYTCHMDVFHFMA G DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 75 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72H 
                 CSLHRSAHNATHATYTCHMDVFHFMA H DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 76 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72I 
                 CSLHRSAHNATHATYTCHMDVFHFMA I DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 77 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72K 
                 CSLHRSAHNATHATYTCHMDVFHFMA K DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 78 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72L 
                 CSLHRSAHNATHATYTCHMDVFHFMA L DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 79 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72M 
                 CSLHRSAHNATHATYTCHMDVFHFMA M DIFSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 80 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72Q 
                 CSLHRSAHNATHATYTCHMDVFHFMA Q DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 81 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72R 
                 CSLHRSAHNATHATYTCHMDVFHFMA R DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 82 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72W 
                 CSLHRSAHNATHATYTCHMDVFHFMA W DIFSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 83 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D72Y 
                 CSLHRSAHNATHATYTCHMDVFHFMA Y DIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 84 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73C 
                 CSLHRSAHNATHATYTCHMDVFHFMAD C IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 85 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73A 
                 CSLHRSAHNATHATYTCHMDVFHFMAD A IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 86 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73E 
                 CSLHRSAHNATHATYTCHMDVFHFMAD E IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 87 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73H 
                 CSLHRSAHNATHATYTCHMDVFHFMAD H IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 88 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73K 
                 CSLHRSAHNATHATYTCHMDVFHFMAD K IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 89 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73R 
                 CSLHRSAHNATHATYTCHMDVFHFMAD R IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 90 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73W 
                 CSLHRSAHNATHATYTCHMDVFHFMAD W IFSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 91 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 D73Y 
                 CSLHRSAHNATHATYTCHMDVFHFMAD Y IFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 92 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 I74H 
                 CSLHRSAHNATHATYTCHMDVFHFMADD H FSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 93 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 I74K 
                 CSLHRSAHNATHATYTCHMDVFHFMADD K FSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 94 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 I74R 
                 CSLHRSAHNATHATYTCHMDVFHFMADD R FSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 95 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 I74W 
                 CSLHRSAHNATHATYTCHMDVFHFMADD W FSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 96 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 L94F 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSF F LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 97 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 L94K 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSF K LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 98 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 L94Q 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSF Q LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 99 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 L94R 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSF R LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 100 
                 QDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAF 
               
               
                 Human IL-21 
                 SCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTC 
               
               
                   
                 PSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS 
               
               
                   
               
               
                 101 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 second chain 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (Heavy chain of 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 anti-PD-1 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 antibody + 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                 linker + 
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                 human IL-21 with 
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                 a knob mutation 
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCL 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW 
               
               
                   
                 SAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHR 
               
               
                   
                 LTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS 
               
               
                   
               
               
                 102 
                 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL 
               
               
                 Anti-PD-1 
                 IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW 
               
               
                 antibody, Light 
                 PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP 
               
               
                 chain 
                 REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD 
               
               
                   
                 YEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 103 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Anti-PD-1 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 antibody, Heavy 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 chain with a hole 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 mutation 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 
               
               
                   
               
               
                 104 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36C 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ C EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 105 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36E 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ E EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 106 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36G 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ G EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 107 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36H 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ H EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 108 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36I 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ I EELKDEATSC 
               
               
                   
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                   
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 109 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36K 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ K EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 110 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36M 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ M EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 111 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36N 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ N EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 112 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36P 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ P EELKDEATSC 
               
               
                   
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                   
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 113 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36Q 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ Q EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 114 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ R EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 115 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36S 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ S EELKDEATSC 
               
               
                   
                 SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG 
               
               
                   
                 SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY 
               
               
                   
                 ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA 
               
               
                   
                 GPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 116 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36T 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ T EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 117 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 Y36V 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ V EELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 118 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 E38C 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE C LKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 119 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 E38Y 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE Y LKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 120 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39C 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE C KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 121 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39E 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE E KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 122 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39H 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE H KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 123 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39K 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE K KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 124 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE R KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 125 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39W 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE W KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 126 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L39Y 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE Y KDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 127 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70F 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF F ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 128 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70H 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF H ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 129 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70N 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF N ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 130 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70Q 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF Q ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 131 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70S 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF S ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 132 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70T 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF T ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 133 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70V 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF V ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 134 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70W 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF W ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 135 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70Y 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF Y ADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 136 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71E 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHF M EDDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 137 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71F 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM F DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 138 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71I 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM I DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 139 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71L 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM L DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 140 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71Q 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM Q DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 141 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM R DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 142 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71W 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM W DDIFSVNITDQSGNYSQE 
               
               
                   
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 143 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 A71Y 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFM Y DDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 144 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 I74R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADD R FSVNITDQSGNYSQE 
               
               
                   
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 145 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 I74W 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADD W FSVNITDQSGNYSQE 
               
               
                   
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 146 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L94F 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSF F LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 147 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L94K 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSF K LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 148 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L94Q 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSF Q LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 149 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L94R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSF R LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 150 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 L94Y 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSF Y LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 151 (Ipilimumab 
                 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG 
               
               
                 heavy chain) 
                 LEWVTFISYDGNNKYY 
               
               
                   
                 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPF 
               
               
                   
                 DYWGQGTLVTVSSAS 
               
               
                   
                 TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 
               
               
                   
                 GVHTFPAVLQSSGL 
               
               
                   
                 YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT 
               
               
                   
                 CPPCPAPELLGGPS 
               
               
                   
                 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV 
               
               
                   
                 HNAKTKPREEQYNST 
               
               
                   
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR 
               
               
                   
                 EPQVYTLPPSRDELT 
               
               
                   
                 KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF 
               
               
                   
                 LYSKLTVDKSRWQQ 
               
               
                   
                 GNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 152 (Ipilimumab 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPR 
               
               
                 light chain) 
                 LLIYGAFSRATGIP 
               
               
                   
                 DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI 
               
               
                   
                 KRTVAAPSVFIFP 
               
               
                   
                 PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV 
               
               
                   
                 TEQDSKDSTYSLSSTL 
               
               
                   
                 TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 153 
                 QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA 
               
               
                 (tremelimumab 
                 PGKGLEWVAV IWYDGSNKYY 
               
               
                 heavy chain) 
                 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP 
               
               
                   
                 RGATLYYYYY GMDVWGQGTT 
               
               
                   
                 VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE 
               
               
                   
                 PVTVSWNSGA LTSGVHTFPA 
               
               
                   
                 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD 
               
               
                   
                 KTVERKCCVE CPPCPAPPVA 
               
               
                   
                 GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN 
               
               
                   
                 WYVDGVEVHN AKTKPREEQF 
               
               
                   
                 NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI 
               
               
                   
                 SKTKGQPREP QVYTLPPSRE 
               
               
                   
                 EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP 
               
               
                   
                 MLDSDGSFFL YSKLTVDKSR 
               
               
                   
                 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 
               
               
                   
               
               
                 154 
                 DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP 
               
               
                 (tremelimumab 
                 GKAPKLLIYA ASSLQSGVPS 
               
               
                 light chain) 
                 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP 
               
               
                   
                 GTKVEIKRTV AAPSVFIFPP 
               
               
                   
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
                 ESVTEQDSKD STYSLSSTLT 
               
               
                   
                 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 
               
               
                   
               
               
                 155 
                 CPDLVCYTD A LQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 Y10A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 156 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ A EELKDEATS 
               
               
                 Y36A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 157 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE A LKDEATS 
               
               
                 E38A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 158 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEE A KDEATS 
               
               
                 L39A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 159 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 F67A 
                 CSLHRSAHNATHATYTCHMDV A HFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 160 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 H68A 
                 CSLHRSAHNATHATYTCHMDVF A FMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 161 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 I74A 
                 CSLHRSAHNATHATYTCHMDVFHFMADD A FSVNITDQSGNYSQE 
               
               
                 (IL-21Rα mutein) 
                 CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL 
               
               
                   
                 QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV 
               
               
                   
                 RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 162 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 L94A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSF A LAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 163 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 P126A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYED A AFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 164 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 Y129A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAF A MLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 165 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 M130A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY A LKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 166 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 K134A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG A LQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 167 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 S189A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPG A SYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 168 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 S190A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGS A YQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 169 
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                 Y191A 
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                 (IL-21Rα mutein) 
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSS A QGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 170 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 Y10A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTD A LQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 171 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 Y36A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQ A EELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 172 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 E38A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYE A LKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 173 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 L39A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEE A KDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 174 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 F67A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDV A HFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 175 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 H68A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVF A FM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 176 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 I74A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADD A FSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 177 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 L94A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21 Ra mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSF A LAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 178 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 P126A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYED A AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 179 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 Y129A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAF A MLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 180 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 M130A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFY A LKGKLQYELQYRNRGDPWAVSPRRKLISVDSR 
               
               
                   
                 SVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQ 
               
               
                   
                 SEELKE 
               
               
                   
               
               
                 181 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 K134A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKG A LQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 182 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 S189A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPG A SYQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 183 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 S190A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGS A YQGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 184 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 Y191A 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (Fc-linker-IL- 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                 21Rα mutein) 
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS 
               
               
                   
                 TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM 
               
               
                   
                 ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI 
               
               
                   
                 SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS 
               
               
                   
                 RSVSLLPLEFRKDSSYELQVRAGPMPGSS A QGTWSEWSDPVIFQT 
               
               
                   
                 QSEELKE 
               
               
                   
               
               
                 185 
                 PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 
               
               
                 IgG1 Fc moiety 
                 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 
               
               
                 (WT) 
                 VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP 
               
               
                   
                 PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL 
               
               
                   
                 SPGK 
               
               
                   
               
               
                 186 
                 ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD 
               
               
                 IgG2 Fc moiety 
                 VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV 
               
               
                 (WT) 
                 HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS 
               
               
                   
                 REEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLD 
               
               
                   
                 SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL 
               
               
                   
                 SLSPGK 
               
               
                   
               
               
                 187 
                 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP 
               
               
                 IgG3 Fc moiety 
                 EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV 
               
               
                 (WT) 
                 VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVL 
               
               
                   
                 TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTL 
               
               
                   
                 PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPM 
               
               
                   
                 LDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL 
               
               
                   
                 SPGK 
               
               
                   
               
               
                 188 
                 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD 
               
               
                 IgG4 Fc moiety 
                 VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL 
               
               
                 (WT) 
                 HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS 
               
               
                   
                 QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                 SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS 
               
               
                   
                 LGK 
               
               
                   
               
               
                 189 
                 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL 
               
               
                 IGHG1 
                 TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK 
               
               
                 (Immunoglobulin 
                 VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT 
               
               
                 heavy constant 
                 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 
               
               
                 gamma 1) with 
                 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR 
               
               
                 ‘LALA’(L234A/ 
                 EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
               
               
                 L235A) mutation 
                 YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH 
               
               
                   
                 YTQKSLSLSPGK 
               
               
                   
               
               
                 190 
                 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL 
               
               
                 IGHG4 
                 TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT 
               
               
                 (Immunoglobulin 
                 KVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEV 
               
               
                 heavy constant 
                 TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV 
               
               
                 gamma 4) with 
                 VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ 
               
               
                 SPLE‘(S228P/L 
                 VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 
               
               
                 235E) mutation 
                 TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT 
               
               
                   
                 QKSLSLSLGK 
               
               
                   
               
               
                 191 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 αPD-1-linker- 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 IL21RαWT 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                   
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                   
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGS 
               
               
                   
                 CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS 
               
               
                   
                 CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC 
               
               
                   
                 GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ 
               
               
                   
                 YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR 
               
               
                   
                 AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 192 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 Y36A + D72E 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQ A EELKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSFLLAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 193 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 Y36A + L94R 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQ A EELKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSF R LAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 194 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 E38A + D72E 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYE A LKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSFLLAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 195 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 E38A + L94K 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYE A LKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSF K LAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 196 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 E38A + L94R 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYE A LKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSF R LAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 197 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 E38R + D72R 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYE R LKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMA R DIFSVNITDQSGNYSQECGSFLLAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 198 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 D72E + L94K 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSF K LAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 199 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG 
               
               
                 D72E + L94R 
                 KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN 
               
               
                 (αPD- 
                 SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP 
               
               
                 1 + linker + IL21Rα 
                 CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES 
               
               
                   
                 KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 
               
               
                   
                 DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV 
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV 
               
               
                   
                 YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY 
               
               
                   
                 KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH 
               
               
                   
                 NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV 
               
               
                   
                 ICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATH 
               
               
                   
                 ATYTCHMDVFHFMA E DIFSVNITDQSGNYSQECGSF R LAESIK 
               
               
                   
                 PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR 
               
               
                   
                 NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP 
               
               
                   
                 MPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 200 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 E38R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE R LKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 201 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70C 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHF C ADDIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 202 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70D 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHF D ADDIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 203 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70G 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHF G ADDIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 204 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 M70R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHF R ADDIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 205 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 D72A 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMA A DIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 206 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 D72E 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMA E DIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 207 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 D72Q 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMA Q DIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 208 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 D72R 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMA R DIFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 209 
                 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG 
               
               
                 D73A 
                 LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE 
               
               
                 (αPD- 
                 DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                 1 + linker + IL21Rα 
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 
               
               
                 Mutein) 
                 VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF 
               
               
                   
                 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV 
               
               
                   
                 DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK 
               
               
                   
                 VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG 
               
               
                   
                 GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE 
               
               
                   
                 ATSCSLHRSAHNATHATYTCHMDVFHFMAD A IFSVNITDQSGNYS 
               
               
                   
                 QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG 
               
               
                   
                 KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL 
               
               
                   
                 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 210 
                 CCGAAATCATGTGACAAAACTCATACTTGTCCTCCATGCCCA 
               
               
                 (Polynucleotide 
                 GCCCCAGAATTGCTGGGGGGACCATCTGTGTTCCTTTTCCCC 
               
               
                 encoding IgG1 
                 CCTAAGCCAAAAGACACTCTGATGATCAGTCGCACTCCTGA 
               
               
                 Fc-IL21Rα 
                 AGTGACCTGCGTCGTGGTAGACGTCTCTCACGAAGATCCCG 
               
               
                 (wildtype)) 
                 AGGTCAAATTTAACTGGTATGTGGATGGCGTGGAAGTTCATA 
               
               
                   
                 ACGCAAAAACCAAACCCCGCGAAGAACAATATAATAGCACA 
               
               
                   
                 TACCGTGTTGTTAGCGTTTTGACAGTCCTTCACCAGGATTGG 
               
               
                   
                 CTCAACGGAAAAGAGTACAAGTGCAAGGTGTCCAATAAAG 
               
               
                   
                 CATTGCCCGCCCCTATAGAGAAGACTATTAGCAAGGCCAAA 
               
               
                   
                 GGTCAGCCCCGGGAGCCTCAGGTGTATACATTGCCTCCCAG 
               
               
                   
                 CCGCGATGAACTCACTAAAAACCAAGTCAGCCTCACATGTC 
               
               
                   
                 TGGTTAAAGGTTTTTACCCCAGCGATATCGCAGTCGAGTGGG 
               
               
                   
                 AATCTAATGGGCAGCCTGAAAATAACTATAAGACAACCCCA 
               
               
                   
                 CCAGTGTTGGATAGCGATGGCAGCTTTTTTCTTTACTCTAAG 
               
               
                   
                 TTGACTGTTGACAAGAGCAGGTGGCAACAAGGCAACGTGT 
               
               
                   
                 TTAGCTGCAGTGTCATGCACGAAGCACTCCACAATCATTACA 
               
               
                   
                 CCCAGAAGAGTCTGAGCTTGTCACCTGGAAAGGGTGGAGG 
               
               
                   
                 CGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCATGTC 
               
               
                   
                 CTGACCTGGTGTGCTACACCGACTACCTGCAGACCGTGATC 
               
               
                   
                 TGCATCCTGGAGATGTGGAACCTGCATCCTTCTACCCTGACA 
               
               
                   
                 CTGACCTGGCAGGACCAGTACGAGGAACTGAAGGACGAGG 
               
               
                   
                 CCACCTCCTGCTCCCTGCACAGATCTGCTCACAACGCCACC 
               
               
                   
                 CACGCTACCTACACCTGTCACATGGACGTGTTCCACTTCATG 
               
               
                   
                 GCCGACGACATCTTTTCTGTGAACATCACCGATCAGTCTGGC 
               
               
                   
                 AACTACTCCCAAGAGTGCGGCTCTTTCCTGCTGGCCGAGTC 
               
               
                   
                 CATCAAGCCTGCTCCTCCTTTCAACGTGACCGTGACCTTCTC 
               
               
                   
                 CGGCCAGTACAACATCTCTTGGCGGTCCGACTACGAGGACC 
               
               
                   
                 CCGCCTTCTACATGCTGAAGGGCAAGCTGCAGTACGAGCTG 
               
               
                   
                 CAGTACCGGAACAGAGGCGACCCTTGGGCCGTGTCCCCTAG 
               
               
                   
                 AAGAAAGCTGATCTCCGTGGACTCCAGATCCGTGTCTCTGC 
               
               
                   
                 TGCCTCTGGAATTCCGGAAGGACTCTAGCTACGAACTGCAA 
               
               
                   
                 GTGCGGGCTGGCCCTATGCCTGGCTCCTCCTACCAGGGAAC 
               
               
                   
                 ATGGTCCGAGTGGAGCGATCCTGTGATCTTCCAGACCCAGT 
               
               
                   
                 CCGAAGAGCTGAAAGAG 
               
               
                   
               
               
                 211 
                 CAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGCAG 
               
               
                 (Polynucleotide 
                 CCAGGCAGGTCCCTGCGGCTGGACTGTAAGGCCTCCGGCA 
               
               
                 encoding αPD-1 
                 TCACCTTTTCTAACTCCGGAATGCATTGGGTGAGGCAGGCT 
               
               
                 IL21RαWT) 
                 CCAGGCAAGGGCCTGGAGTGGGTGGCTGTGATCTGGTACG 
               
               
                   
                 ACGGCAGCAAGCGGTACTATGCCGATTCTGTGAAGGGCAG 
               
               
                   
                 ATTCACAATCTCTCGCGACAACTCCAAGAATACCCTGTTTC 
               
               
                   
                 TGCAGATGAACTCTCTGAGGGCCGAGGATACAGCCGTGTA 
               
               
                   
                 CTATTGCGCTACCAATGACGATTACTGGGGCCAGGGCACAC 
               
               
                   
                 TGGTGACCGTGTCCAGCGCCAGCACAAAGGGACCATCCGT 
               
               
                   
                 GTTCCCACTGGCTCCATGCAGCCGGTCTACATCCGAGAGCA 
               
               
                   
                 CCGCCGCTCTGGGATGTCTGGTGAAGGATTATTTCCCTGAG 
               
               
                   
                 CCAGTGACCGTGAGCTGGAACTCCGGCGCCCTGACATCTG 
               
               
                   
                 GCGTGCACACCTTTCCTGCTGTGCTGCAGTCTTCCGGCCTG 
               
               
                   
                 TACTCCCTGAGCTCTGTGGTGACAGTGCCCTCCAGCTCTCT 
               
               
                   
                 GGGCACCAAGACATATACCTGCAACGTGGACCATAAGCCTT 
               
               
                   
                 CCAATACCAAGGTGGATAAGAGAGTGGAGAGCAAGTACGG 
               
               
                   
                 ACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTCTGGGAG 
               
               
                   
                 GACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACC 
               
               
                   
                 CTGATGATCAGCCGCACACCTGAGGTGACCTGCGTGGTGG 
               
               
                   
                 TGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTG 
               
               
                   
                 GTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACCAAG 
               
               
                   
                 CCTAGAGAGGAGCAGTTTAACTCCACATACCGCGTGGTGA 
               
               
                   
                 GCGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAA 
               
               
                   
                 GGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCATCCA 
               
               
                   
                 GCATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCTAG 
               
               
                   
                 GGAGCCACAGGTGTACACCCTGCCCCCTTCTCAGGAGGAG 
               
               
                   
                 ATGACAAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGG 
               
               
                   
                 GCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAAT 
               
               
                   
                 GGCCAGCCCGAGAACAATTACAAGACCACACCACCCGTGC 
               
               
                   
                 TGGACTCCGATGGCAGCTTCTTTCTGGTCTCCAGGCTGACA 
               
               
                   
                 GTGGATAAGAGCCGGTGGCAGGAGGGCAACGTGTTTTCTT 
               
               
                   
                 GTTCCGTGATGCACGAGGCTCTGCACAATCATTACACCCAG 
               
               
                   
                 AAGAGCCTGTCTCTGTCCCTGGGCGGTGGCGGTGGCTCTG 
               
               
                   
                 GCGGAGGTGGCTCAGGTGGCGGCGGATCCTGTCCTGATCT 
               
               
                   
                 CGTGTGCTATACCGACTACCTCCAGACCGTTATTTGTATCCT 
               
               
                   
                 TGAGATGTGGAATTTGCACCCATCAACACTGACTCTGACTT 
               
               
                   
                 GGCAGGATCAATACGAGGAGCTGAAAGACGAGGCCACATC 
               
               
                   
                 CTGCTCCTTGCATCGATCAGCACACAACGCCACTCATGCAA 
               
               
                   
                 CATACACTTGCCATATGGATGTGTTCCACTTCATGGCAGATG 
               
               
                   
                 ATATTTTTTCAGTTAACATTACAGATCAATCCGGCAACTATT 
               
               
                   
                 CACAGGAATGTGGCTCTTTTCTTCTGGCAGAATCAATAAAG 
               
               
                   
                 CCCGCACCTCCTTTCAACGTGACTGTCACCTTCTCAGGACA 
               
               
                   
                 ATATAATATCAGCTGGCGATCTGACTATGAGGACCCTGCCTT 
               
               
                   
                 TTACATGCTGAAAGGCAAGCTCCAATACGAACTTCAATATC 
               
               
                   
                 GTAATAGGGGGGACCCATGGGCCGTCAGTCCTCGACGGAA 
               
               
                   
                 GCTGATATCCGTGGACTCTAGAAGTGTCTCTCTCTTGCCCCT 
               
               
                   
                 CGAATTTAGGAAAGACTCATCCTACGAGCTTCAAGTTCGGG 
               
               
                   
                 CAGGTCCCATGCCCGGCTCAAGCTATCAGGGGACATGGAG 
               
               
                   
                 CGAGTGGTCCGACCCAGTAATTTTCCAAACCCAAAGCGAG 
               
               
                   
                 GAATTGAAAGAG 
               
               
                   
               
               
                 212 
                 GGGGS 
               
               
                 (GGGGS) 1   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 213 
                 GGGGSGGGGS 
               
               
                 (GGGGS) 2   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 214 
                 GGGGSGGGGSGGGGS 
               
               
                 (GGGGS) 3   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 215 
                 GGGGSGGGGSGGGGSGGGGS 
               
               
                 (GGGGS) 4   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 216 
                 GGGGGG 
               
               
                 (Gly) 6   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 217 
                 GGGGGGGG 
               
               
                 (Gly) 8   
                   
               
               
                 Flexible Linker 
                   
               
               
                   
               
               
                 218 
                 EAAAK 
               
               
                 (EAAAK) 1   
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 219 
                 EAAAKEAAAK 
               
               
                 (EAAAK) 2   
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 220 
                 EAAAKEAAAKEAAAK 
               
               
                 (EAAAK) 3   
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 221 
                 AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAA 
               
               
                 A(EAAAK) 4 ALE 
                 KEAAAKA 
               
               
                 A(EAAAK) 4 A 
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 222 
                 PAPAP 
               
               
                 PAPAP 
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 223 
                 AEAAAKEAAAKA 
               
               
                 AEAAAKEAAAKA 
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 224 
                 (AP)n, (n = 5-15) 
               
               
                 (Ala-Pro)n 
                   
               
               
                 (10-33 aa) 
                   
               
               
                 Rigid Linker 
                   
               
               
                   
               
               
                 225 
                 EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG 
               
               
                 Tiragolumab 
                 LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP 
               
               
                 Heavy chain 
                 EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL 
               
               
                   
                 APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                   
                 QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 
               
               
                   
                 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
               
               
                   
                 DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                 MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                 SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 226 
                 DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQK 
               
               
                 Tiragolumab light 
                 PGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY 
               
               
                 chain 
                 YCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV 
               
               
                   
                 CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS 
               
               
                   
                 TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 227 
                 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG 
               
               
                 Relatlimab Heavy 
                 LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT 
               
               
                 chain 
                 AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                   
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
               
               
                   
                 LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP 
               
               
                   
                 PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
               
               
                   
                 FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
               
               
                   
                 EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV 
               
               
                   
                 SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR 
               
               
                   
                 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 228 
                 EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLI 
               
               
                 Relatlimab Light 
                 YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP 
               
               
                 chain 
                 LTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR 
               
               
                   
                 EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE 
               
               
                   
                 KHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 229 
                 
                   QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG 
                 
               
               
                 Second Chain 
                 
                   LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE 
                 
               
               
                 (Heavy chain of 
                 
                   DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK 
                 
               
               
                 anti-CTLA-4 
                 
                   STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL 
                 
               
               
                 antibody + linker + 
                 
                   YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT 
                 
               
               
                 human IL-21) 
                 
                   CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 
                 
               
               
                 with knob 
                 
                   VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 
                 
               
               
                 mutation 
                 
                   GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN 
                 
               
               
                   
                 
                   QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL 
                 
               
               
                   
                 
                   YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGG 
                 
               
               
                   
                 
                   SGGGGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPE 
                 
               
               
                   
                 
                   DVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTN 
                 
               
               
                   
                 
                   AGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH 
                 
               
               
                   
                 
                   GSEDS 
                 
               
               
                   
               
               
                 230 
                 
                   QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG 
                 
               
               
                 αCTLA-4 + 
                 
                   LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE 
                 
               
               
                 linker + IL21Rα 
                 
                   DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK 
                 
               
               
                 WT 
                 
                   STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL 
                 
               
               
                   
                 
                   YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT 
                 
               
               
                   
                 
                   CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 
                 
               
               
                   
                 
                   VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 
                 
               
               
                   
                 
                   GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN 
                 
               
               
                   
                 
                   QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV 
                 
               
               
                   
                 
                   SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS 
                 
               
               
                   
                 
                   GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ 
                 
               
               
                   
                 
                   YEELKD E ATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNIT 
                 
               
               
                   
                 
                   DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP 
                 
               
               
                   
                 
                   AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF 
                 
               
               
                   
                 
                   RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
                 
               
               
                   
               
               
                 231 
                 
                   QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG 
                 
               
               
                 M70D 
                 
                   LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE 
                 
               
               
                 (αCTLA-4 + 
                 
                   DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK 
                 
               
               
                 linker + IL21Rα 
                 
                   STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL 
                 
               
               
                 mutein) 
                 
                   YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT 
                 
               
               
                   
                 
                   CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 
                 
               
               
                   
                 
                   VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 
                 
               
               
                   
                 
                   GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN 
                 
               
               
                   
                 
                   QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV 
                 
               
               
                   
                 
                   SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS 
                 
               
               
                   
                 
                   GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ 
                 
               
               
                   
                 
                   YEELKD E ATSCSLHRSAHNATHATYTCHMDVFHFDADDIFSVNIT 
                 
               
               
                   
                 
                   DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP 
                 
               
               
                   
                 
                   AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF 
                 
               
               
                   
                 
                   RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
                 
               
               
                   
               
               
                 232 
                 
                   QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG 
                 
               
               
                 D72A 
                 
                   LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE 
                 
               
               
                 (αCTLA-4 + 
                 
                   DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK 
                 
               
               
                 linker + IL21Rα 
                 
                   STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL 
                 
               
               
                 mutein) 
                 
                   YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT 
                 
               
               
                   
                 
                   CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE 
                 
               
               
                   
                 
                   VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 
                 
               
               
                   
                 
                   GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN 
                 
               
               
                   
                 
                   QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV 
                 
               
               
                   
                 
                   SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS 
                 
               
               
                   
                 
                   GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ 
                 
               
               
                   
                 
                   YEELKD E ATSCSLHRSAHNATHATYTCHMDVFHFMAADIFSVNIT 
                 
               
               
                   
                 
                   DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP 
                 
               
               
                   
                 
                   AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF 
                 
               
               
                   
                 
                   RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
                 
               
               
                   
               
               
                 233 
                 
                   EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG 
                 
               
               
                 Second Chain 
                 
                   LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP 
                 
               
               
                 (Heavy chain of 
                 
                   EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL 
                 
               
               
                 anti-TIGIT 
                 
                   APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
                 
               
               
                 antibody + 
                 
                   QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 
                 
               
               
                 linker + human 
                 
                   DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
                 
               
               
                 IL-21) with knob 
                 
                   HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
                 
               
               
                 mutation 
                 
                   DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
                 
               
               
                   
                 
                   MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD 
                 
               
               
                   
                 
                   GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 
                 
               
               
                   
                 
                   GGGGSGGGGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEF 
                 
               
               
                   
                 
                   LPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRK 
                 
               
               
                   
                 
                   PPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHL 
                 
               
               
                   
                 
                   SSRTHGSEDS 
                 
               
               
                   
               
               
                 234 
                 
                   EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG 
                 
               
               
                 αTIGIT + 
                 
                   LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP 
                 
               
               
                 linker + IL21Rα 
                 
                   EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL 
                 
               
               
                 WT 
                 
                   APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
                 
               
               
                   
                 
                   QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 
                 
               
               
                   
                 
                   DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
                 
               
               
                   
                 
                   HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
                 
               
               
                   
                 
                   DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
                 
               
               
                   
                 
                   MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
                 
               
               
                   
                 
                   SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG 
                 
               
               
                   
                 
                   GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT 
                 
               
               
                   
                 
                   WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIF 
                 
               
               
                   
                 
                   SVNIT D QSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD 
                 
               
               
                   
                 
                   YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL 
                 
               
               
                   
                 
                   PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK 
                 
               
               
                   
                 
                   E 
                 
               
               
                   
               
               
                 235 
                 EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG 
               
               
                 M70D 
                 LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP 
               
               
                 (αTIGIT + 
                 EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL 
               
               
                 linker + IL21Rα 
                 APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 
               
               
                   
                 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
               
               
                   
                 DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                 MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                 SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG 
               
               
                   
                 GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT 
               
               
                   
                 WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFDADDIF 
               
               
                   
                 SVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD 
               
               
                   
                 YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL 
               
               
                   
                 PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK 
               
               
                   
                 E 
               
               
                   
               
               
                 236 
                 EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG 
               
               
                 D72A 
                 LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP 
               
               
                 (αTIGIT + 
                 EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL 
               
               
                 linker + IL21Rα 
                 APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 
               
               
                 Mutein) 
                 QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 
               
               
                   
                 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS 
               
               
                   
                 HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ 
               
               
                   
                 DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 
               
               
                   
                 MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                 SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG 
               
               
                   
                 GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT 
               
               
                   
                 WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAADIF 
               
               
                   
                 SVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD 
               
               
                   
                 YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL 
               
               
                   
                 PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK 
               
               
                   
                 E 
               
               
                   
               
               
                 237 
                 
                   QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG 
                 
               
               
                 Second Chain 
                 
                   LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT 
                 
               
               
                 (Heavy chain of 
                 
                   AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS 
                 
               
               
                 anti-LAG-3 
                 
                   RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
                 
               
               
                 antibody + linker + 
                 
                   LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP 
                 
               
               
                 human IL-21) 
                 
                   PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
                 
               
               
                 with knob 
                 
                   FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
                 
               
               
                 mutation 
                 
                   EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV 
                 
               
               
                   
                 
                   SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR 
                 
               
               
                   
                 
                   LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG 
                 
               
               
                   
                 
                   GGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVE 
                 
               
               
                   
                 
                   TNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGR 
                 
               
               
                   
                 
                   RQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSE 
                 
               
               
                   
                 
                   DS 
                 
               
               
                   
               
               
                 238 
                 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG 
               
               
                 αLAG-3 + 
                 LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT 
               
               
                 linker + IL21Rα 
                 AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                 WT 
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
               
               
                   
                 LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP 
               
               
                   
                 PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
               
               
                   
                 FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
               
               
                   
                 EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV 
               
               
                   
                 SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR 
               
               
                   
                 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG 
               
               
                   
                 GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE 
               
               
                   
                 ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQ 
               
               
                   
                 SGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY 
               
               
                   
                 MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD 
               
               
                   
                 SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
               
               
                   
               
               
                 239 
                 
                   QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG 
                 
               
               
                 M70D 
                 
                   LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT 
                 
               
               
                 (αLAG-3 + 
                 
                   AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS 
                 
               
               
                 inker + IL21Rα 
                 
                   RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
                 
               
               
                 Mutein) 
                 
                   LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP 
                 
               
               
                   
                 
                   PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
                 
               
               
                   
                 
                   FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
                 
               
               
                   
                 
                   EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV 
                 
               
               
                   
                 
                   SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR 
                 
               
               
                   
                 
                   LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG 
                 
               
               
                   
                 
                   GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE 
                 
               
               
                   
                 
                   ELKDEATSCSLHRSAHNATHATYTCHMDVFHFDADDIFSVNITDQS 
                 
               
               
                   
                 
                   GNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY 
                 
               
               
                   
                 
                   MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD 
                 
               
               
                   
                 
                   SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE 
                 
               
               
                   
               
               
                 240 
                 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG 
               
               
                 D72A 
                 LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT 
               
               
                 (αLAG-3 + 
                 AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS 
               
               
                 linker + IL21Rα 
                 RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
               
               
                 Mutein) 
                 LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP 
               
               
                   
                 PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ 
               
               
                   
                 FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
               
               
                   
                 EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV 
               
               
                   
                 SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR 
               
               
                   
                 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG 
               
               
                   
                 GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE 
               
               
                   
                 ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAADIFSVNITDQ 
               
               
                   
                 SGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY 
               
               
                   
                 MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD 
               
               
                   
                 SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE