Office Action 3: Notice of Allowance (2025-04-08)

CLAIMS:
Amendment filed March 14, 2025 Reply to Office Action dated January 22, 2025 AMENDMENTS TO THE CLAIMS:

1\. (Currently Amended) A D Domain target binding domain polypeptide that specifically binds CD 123 and comprises thean amino acid sequence of SEQ ID NO: 14.

2\. (Original) A polypeptide comprising the D domain of claim 1 fused to a heterologous polypeptide.

3\. (Currently Amended) The polypeptide of claim 2, wherein the heterologous polypeptide comprises:\[\[.\]\] (a) a full-length antibody or an antibody fragment;(b) an Fe domain;
(c) a transmembrane domain;
(d) a membrane associating domain;
(e) human serum albumin or a fragment thereof;
(f) AFP or a fragment thereof;
(g) AFP p26 or a fragment thereof; or (h) the extracellular domain of a receptor or a fragment thereof.
4\. (Original) The polypeptide of claim 2, which is labeled or conjugated to a therapeutic or cytotoxic agent.

5\. (Original) A chimeric antigen receptor (CAR) which comprises a target binding domain comprising the D domain that specifically binds CD 123 of claim 1.

6\. (Previously Presented) The CAR of claim 5, further comprising a transmembrane domain and an intracellular signaling domain.

7\. (Original) The CAR of claim 6, wherein (a) the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain;(b) the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof; and/or

(c) the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB,OX40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1),
CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

8\. (Previously Presented) The CAR of claim 5 comprising the amino acid sequence of SEQ ID NO: 67.

9\. (Previously Presented) An adapter comprising (a) the D domain target binding domain that specifically binds CD123 of claim 1, and (b) an antigenic determinant (AD).

10\. (Previously Presented) The adapter of claim 9, wherein the AD comprises an AFP p26 polypeptide.

11\. (Previously Presented) The adapter of claim 10, wherein the AFP p26 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37- 43 and 44.

12\. (Previously Presented) The adapter of claim 9, wherein the adapter comprises a single D domain that specifically binds CD123 or two D domains that specifically bind CD123.

13\. (Original) An isolated polynucleotide encoding the polypeptide of claim 2.

14\. (Original) A method of producing the polypeptide of claim 2, comprising culturing a host cell comprising a polynucleotide encoding the polypeptide under suitable conditions to produce the polypeptide.

15\. (Original) An isolated polynucleotide encoding the CAR of claim 5.

16\. (Original) A lentiviral vector comprising the polynucleotide of claim 15.

17\. (Original) A host cell comprising the polynucleotide of claim 15.

18\. (Original) A cell engineered to express the CAR of claim 5.

19\. (Original) The cell of claim 18, which is a T cell or a natural killer (NK) cell.

20\. (Previously Presented) A pharmaceutical composition comprising the polypeptide of claim 2 and a pharmaceutically acceptable excipient.

21\. (Original) A pharmaceutical composition comprising a cell expressing the CAR of claim 5 and a pharmaceutically acceptable excipient.

22\. (Original) The pharmaceutical composition of claim 21, wherein the cell is a T cell or a natural killer (NK) cell.

23\. (Currently Amended) A method of delivering an immune response to a target cell or killing a target cell comprising;(a) contacting a composition comprising the target cell with a cell expressing a chimeric antigen receptor (CAR) comprising (i) the D domain that binds to CD 123 of claim 1, (ii) a transmembrane domain, and (iii) an intracellular signaling domain;
(b) contacting a composition comprising the target cell with an adapter, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain,
and (iii) an intracellular signaling domain; and (b) the adapter comprises (i) the D domain that binds to CD 123 of claim 1 and (ii) AFP p26 AD;
(c) contacting a composition comprising the target cell with a cell expressing a CAR,wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular signaling domain; and (b) the composition comprising the target cell further comprises an adapter comprising (i) the D domain that binds to CD 123 of claim 1 and (ii) an AFP p26 AD; or (d) contacting a composition comprising the target cell with a cell expressing a CAR and an adapter, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular signaling domain; and (b) the adapter comprises (i) the D domain that binds to CD 123 of claim 1 and (ii) an AFP p26 AD.

24\. (Previously Presented) The method of claim 23, wherein (a) the transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain;

Reply to Office Action dated January 22, 2025 (b) the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof;(c) the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB,OX40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1),
CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof;
(d) the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44;
(e) the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD;
(f) the ADBD that binds to AFP p26 AD comprises a D domain comprising the amino acid sequence of SEQ ID NO: 73, 70-72 or 92-94;
(g) the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 68 or 69;
(h) the cell expressing the CAR is an immune cell,
(i) the cell expressing the CAR is a T cell or a natural killer (NK) cell;
(j) the contacting occurs in a human subject;
(k) the target cell is a cancer cell; and/or (1) the target cell is an acute myeloid leukemia (AML) cell or a high-risk myelodysplastic syndrome cell.

25\. (Previously Presented) A method of delivering an immune response to a target cell, killing a target cell or treating cancer in a patient comprising:(a) administering to the patient a cell expressing the chimeric antigen receptor (CAR) comprising (i) the D domain that binds to CD123 of claim 1, (ii) a transmembrane domain, and (iii) an intracellular domain; or (b) administering to the patient an adapter comprising (i) the D domain that binds to CD 123 of claim 1 and (ii) an AFP p26 AD.

26\. (Previously Presented) The method of claim 25, wherein

(a) the patient receiving the adapter has been administered a cell expressing a CAR,
wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain,
and (iii) an intracellular signaling domain;
(b) the patient receiving the adapter comprises a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular signaling domain; or (c) the method of (b) further comprises administering a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

27\. (Previously Presented) The method of claim 26, wherein (a) the transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain;(b) the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof;
(c) the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB,OX40, CD30, CD40, PD1, lymphocyte function-associated antigen- 1 (LFA-1),
CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof;
(d) the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44;
(e) the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD;
(f) the ADBD that binds to AFP p26 AD comprises a D domain comprising the amino acid sequence of SEQ ID NO: 73, 70-72 or 92-94;

Amendment filed March 14, 2025 Reply to Office Action dated January 22, 2025 (g) the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 68 or 69;(h) the cell expressing the CAR is an immune cell,
(i) the cell expressing the CAR is a T cell or a natural killer (NK) cell;
(j) the cell expressing the CAR is an autologous immune cell;
(k) the cancer is a hematological cancer; and/or (1) the cancer is acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome.

28-30. (Cancelled)2929

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
The instant application, filed 10/13/2023, is a continuation of application 18/055,096, filed 11/14/2022, and claims domestic benefit to US provisional application 63/279,489, filed 11/15/2021.
Status of Application, Amendments, and/or Claims
Applicant’s amendment after final submitted on 03/14/2025 is acknowledged and the amendment is entered. Claims 1, 3, and 23 are amended and claims 28-30 are cancelled. Claims 1-27 are pending and found to be allowable for the reasons discussed below.
Withdrawn Objections/Rejections
In the office action of 01/22/2025, claims 1-27 and 29-30 were rejected under 35 USC 112(a) written description. Applicant’s amendment to the independent claim to recite that the amino acid sequence is SEQ ID NO: 14 has overcome the rejection and the rejection is withdrawn.
Reasons for Allowance
Claims 1-27 are allowed.
The following is an examiner’s statement of reasons for allowance: The instant claims are drawn to a D Domain target binding domain polypeptide that binds CD123 comprising the amino acid sequence of SEQ ID NO: 14. In searches of the prior art, no D Domain binding polypeptides that specifically bind CD123 were identified with an amino acid structure identical to instant SEQ ID NO: 14. Additionally, no suggestion was identified in the art that would have encouraged an ordinarily skilled artisan to alter known D domains to arrive at instant SEQ ID NO: 14 with a reasonable expectation that the resulting D domain would be specific for CD123. As such, the claimed sequence was also found to be novel and non-obvious. The following is considered to be the closest prior art:
WO 2016/164308 A1 (Lafleur, D.W. and D.M. Hilbert) 13 OCT 2016
WO’308 is drawn to de novo binding domain containing polypeptides (DBDpp) that specifically bind a target of interest. WO’308 teaches nucleic acids encoding the polypeptides as well as vectors, and host cells containing the nucleic acids. WO’308 also teaches libraries of DBDpp as well as methods of producing and screening the libraries and methods of using the DBDpp (abstract).
WO’308 discloses SEQ ID NO: 1 which is a non-naturally occurring and targetless antiparallel three-helical bundle that can be used as a reference scaffold platform for producing de novo binding domain (DBD) containing polypeptides (DBDpp) that bind to a target of interest.  WO’308 further discloses that the scaffold can be used to create libraries which can be screened for DBDpp that have desired functional and/or biological activity (page 52, [0151]).
WO’308 discloses the generation and selection of CD123 targeting DBDpp based off of the reference sequence of SEQ ID NO: 1 which was modified in a plurality of positions resulting in a library of DBDpp corresponding to the genus SEQ ID NOs: 2, 4, and 6. These sequences represent numerous DBDpp exhibiting specificity for CD123 (page 199, [0520]-[0522]). WO’308 further de-immunized a species of SEQ ID NO: 4 to generate SEQ ID NO: 135, a DBDpp with specificity for CD123 (page 200, [0525]; page 149, SEQ ID NO: 135). WO’308, SEQ ID NO: 135 is identical to the parent D domain of the instant application (instant SEQ ID NO: 5) but differs from instantly claimed SEQ ID NO: 14 by 5 substitutions, as shown in the ABSS alignment below:
Instant SEQ ID NO: 14 aligned with WO’308, SEQ ID NO: 135
PNG
media_image1.png
183
624
media_image1.png
Greyscale
As shown in the alignments above, the claimed sequence differs from WO’308, SEQ ID NO: 135 with the following substitutions: G8S, L21Q, K48E, N50S, and I59L.
WO’308, SEQ ID NOs: 2, 4, and 6 suggest that the amino acids in positions 8 and 59 could be substituted in SEQ ID NO: 135 and result in a DBDpp that maintains specificity for CD123 (pages 207-208, claim 21). WO’308, however, does not provide a reasonable expectation that the substitution of the amino acids in positions 21, 48, or 50 would still result in a binding polypeptide specific for CD123. WO’308 identifies these positions as positions that could be modified in SEQ ID NO: 1, however, SEQ ID NO: 1 is considered by the reference to be targetless and, therefore, there is no suggestion that modifying these amino acids as claimed would maintain specificity for CD123. Additionally, WO’308 does not disclose CD123 binding polypeptides that comprise substitutions in positions 21, 48, or 50.
Based on the nature of binding polypeptide functionality and the sensitivity of the binding polypeptides to variation in the amino acid sequences, it would not have been obvious or predictable to one of ordinary skill in the art prior to the effective filing date of the claimed invention to have modified the teachings of WO’308 to arrive at the exact binding polypeptide structure of the instantly claimed invention without undergoing a significant amount of trial and error experimentation to ensure specificity for CD123. Therefore, the instantly claimed sequence was found to be free of, and non-obvious in view of, the prior art.
Conclusions
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”

APPLICANT REBUTTAL: REMARKSClaims 1-30 were pending prior to entry of the abovementioned amendments. Claims 28-30 are herein canceled. Claims 1, 3, and 23 are herein amended. No new matter is added by any of these amendments. Accordingly, upon entry, claims 1-27 will be pending.Reconsideration and allowance of this application is respectfully requested in light of the abovementioned amendments and the following remarks.35 U.S.C. § 112 Claims 1-27 and 29-30 stand rejected under 35 U.S.C. §112(a) for allegedly failing to comply with the written description requirement.As a preliminary matter, Applicant has cancelled claims 29-30, rendering their rejection moot.Further, without conceding to the basis of the rejection and solely expedite prosecution, Applicant has amended claim 1 to recite "[a] D Domain target binding domain polypeptide that specifically binds CD123 and comprises the amino acid sequence of SEQ ID NO: 14." Thereby, claim 1 incorporates the subject matter of claim 28, which the Examiner indicated would be allowable if rewritten in independent form.Accordingly, Applicant respectfully requests that the written description rejection under § 112(a) of claim 1 be withdrawn. Further, each of claims 2-27 depends, directly or indirectly, from claim 1 and thus include all elements of claim 1. Therefore, for at least the reasons discussed above with respect to independent claim 1, Applicant submits that claims 2-27 comply with the written description requirement. Accordingly, Applicant respectfully requests that the written description rejection under § 112(a) of claims 2-27 be withdrawn.It shall be understood herein that any instance in which Applicant has addressed certain comments set forth by the Office shall not be construed as a concession to other comments or arguments advanced by the Office. Any circumstance in which Applicant has amended or canceled a claim also does not mean that Applicant concedes to the arguments or positions advanced by the Office with respect to that claim or other claims pending herein.CONCLUSIONIn view of the amendments and remarks above, favorable reconsideration of the application is respectfully requested and Applicant respectfully requests the Examiner to move this application to issuance. Should the Examiner have any comments or questions, the Examiner is respectfully encouraged to contact the undersigned.The Commissioner is authorized to charge any additional fees which may be required, including petition fees and extension of time fees, to Deposit Account No. 23-2415 (Docket No. 48104-708.301).Respectfully submitted, WILSON SONSINI GOODRICH & ROSATIDate: March 14, 2025


CITED REFERENCE SUMMARIES: Ref WO2016164308A1: Claims:
A method for purifying a target of interest comprising,
contacting a sample comprising a target of interest with a composition comprising: a polypeptide agent attached to a solid support, wherein the polypeptide agent has an amino acid sequence comprising:
MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESEL
QAYKGKG PEVEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQAYr<HN (SEQ ID NO:4), wherein the polypeptide has an amino acid sequence that differs from SEQ ID
wherein the polypeptide specifically binds the target of interest,
wherein the polypeptide's specific binding to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: 1 to the target of interest; the contacting performed under conditions that permit binding of the composition to the target of interest; and
removing a portion of the sample that is not bound to the composition.
The method of Claim 1, further comprising dissociating the composition from the target of interest and recovering the target of interest.
The method of Claim 1, wherein the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose.
The method of Claim 1, wherein the polypeptide agent is coupled to the solid support through non-covalent association.
The method of Claim 1, wherein the polypeptide agent is coupled to the solid support through covalent bonding.
The method of Claim 1, wherein the polypeptide agent of the composition further comprises a peptide tag, wherein the peptide tag comprises a hexahistidine moiety or a FLAG tag.
The method of Claim I, wherein the polypeptide agent of the composition further comprises a streptavidin moiety.
The method of Claim 1, wherein the solid support comprises a bead, and the composition is suitable for use in affinity chromatography to purify the target of interest.
A method according to of any one of Claims 1 to 8, wherein the target of interest can be eluted from the composition.
The method of Claim 1, wherein a nucleic acid molecule encoding the polypeptide is packaged in an expression vector that is used to transduce a cell line to cause the cell line to express the polypeptide.
contacting a sample comprising a target of interest with a composition comprising: a virus-like particle coupled to a solid support, wherein the virus-like particle expresses a polypeptide as a membrane protein, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,
wherein no cysteine or proline residues are substituted into any of SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein the polypeptide has an amino acid sequence that differs from SEQ ID
wherein the polypeptide's specific binding to the target of interest is greater than binding of a polypeptide according to SEQ ID NO: 1 to the target of interest; and the contacting performed under conditions that permit binding of the composition to the target of interest; and
The method of Claim 11, wherein the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose.
The method of Claim 11, wherein the polypeptide of the composition further comprises a peptide tag, wherein the peptide tag comprises a hexahistidine moiety or a FLAG tag.
The method of Claim 11, wherein the portion of the sample that is not bound to the composition is discarded.
The method of Claim 11, wherein the portion of the sample that is not bound to the composition is contacted with the composition a second time to capture additional target of interest.
The method of Claim 15, further comprising contacting the portion of the sample that is not bound to the composition with an antibody directed against the polypeptide of the composition, the antibody being generated from membrane bound virus-like particles (VLP) expressing the polypeptide released from a mammalian cell is engineered to express a fusion protein comprising the polypeptide fused to a chimeric antigen receptor (CAR), the fusion protein being expressed on the generated VLPs,
wherein the antibodies are suitable for use in an assay to detect residual polypeptides detached from the solid support.
A method for removing one or more contaminants from a sample comprising a target of interest comprising,
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID N0 5, and SEQ ID NO:6, 

 wherein the polypeptide has an amino acid sequence that differs from SEQ ID
wherein the polypeptide specifically binds one or more contaminants to be removed from a sample comprising the target of interest,
wherein the polypeptide's specific binding to one or more contaminants is greater than binding of a polypeptide according to SEQ ID NO: l to the one or more contaminants; and
the contacting performed under conditions that permit binding of the composition to the one or more contaminants; and
collecting a portion of the sample that is not bound to the composition.
The method of Claim 17, wherein the polypeptide of the composition further comprises a peptide tag, wherein the peptide tag comprises a hexahistindine moiety or a FLAG tag.
The method of Claim 17, wherein the solid support comprises a bead, a glass slide, a chip, a gelatin, or an agarose and the virus-like particles is coupled to the solid support through non- covalent association.
The method of Claim 17, wherein the portion of the sample that collected is contacted with the composition a second time to remove additional contaminants from the sample.
An affinity resin comprising a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:
MGSWX5X6FKX9X10LAX13IKX16X17LEALGGSEAELAX30
FEX33X34IAX37FEX40X41LQX44YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);
MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AY KGKGNPEVEALXsyXssEAXgiAEX^XesELXggAYRHN (SEQ ID NO:3);
MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEKEIAAFESELQAYK GKGNPEVEX55LRX58X59AAX62IRX65X66LQAYRHN (SEQ ID NO:4); 

 MGSWX 5 X 6 FKX 9 XioLA i 3 lKXi 6 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 4 oE LX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AA X 62 IRX 65 X 66 LQAYRHN (SEQ ID NO: 5);
MGSWX5EFX 8 X9RLXi 2 AIXi5Xi6RLXi9ALGGSEAELAX3oFEX33X34lAX 37 FEX4oX 4iLQX 44 YKGKGNPEVEALX 57 X 58 EAX 6 iAIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);
MGSWX5X6FKX9XioLAXi3lKXi 6 Xi 7 LEALZiEAELAX28FEX3iX32lAX35FEX 3 8 X 39 LQX 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);
MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 N PEVEALX 52 X 53 EAX 5 cAIX 59 X 6 oELX 63 AYRHN (SEQ ID NO:8);
MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALZ 1 EAELAAF
EKEIAAFESELQAYZ2NPEVEX50LRX53X54AAX57IRX60X61LQAYRHN (SEQ ID NO: 9);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKX 16 X 17 LEALZiEAELAAFX 3 oX 3 iEIX 34 AFX 37 X 38 EL X41AYZ2 PEVEX50LRX53X54AAX57IRX60X61 LQAYRHN (SEQ ID NO: 10); and
X 9 RLXi 2 AIXi 5 Xi 6 RLXi 9 ALZiEAELAX 28 FEX 3 iX 32 lAX 35 FEX 38 X 39 LQX 42 YZ 2 PEVEAL X52X53EAX56AIX59X60ELX63AY HN (SEQ ID NO: 11); and combinations thereof;
wherein X n is a natural or non-natural amino acid; wherein each X n is the same or different natural or non-natural amino acid; and/or
wherein Zi and/or Z 2 is 2 to 30 natural or non-natural amino acids;
and wherein the amino acid sequence is not SEQ ID NO: 1.
The affinity resin of claim 21, wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: 1 by an amino acid substitution at one or more residues.
The affinity resin of claim 22, wherein the amino acid substitution at one or more residues comprises a conservative substitution.
The affinity resin of claim 22, wherein the amino acid substitution at one or more residues comprises a non-conservative substitution.
An affinity resin according to any one of claims 22-24, wherein the amino acid substitution at one or more residues comprises a substitution at a solvent accessible residue
An affinity resin according to any one of claims 22-24, wherein the amino acid substitution at one or more residues comprises a substitution at a solvent inaccessible residue.
The affinity resin of claim 21, wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: 1 by an amino acid deletion at one or more residues.
A method of making an affinity resin comprising a step of:
attaching to a solid support a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:
MGS WX5X6FKX9X1 oL AX, 3IKX, 6 X, 7LEALGGSEAEL AX 30
FEX 33 X 34 IAX 37 FEX 40 X 41 LQX 44 YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);
MGSWAEFKQ LAAlKTRLEALGGSEAELAAFX 32 X 33 ETX 36 AFX 39 X 4 oELX 43 AY KGKGNPEVEALX 57 X 58 EAX 6 iAIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);
MGSWX 5 EFX 8 X 9 RLX 12 AIX 15 X 16 RLX 19 ALGGSEAELAAFEI EIAAFESELQAYK GKGNPE EX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQ AYRUN (SEQ ID NO:4);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKXi 6 Xi 7 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 4 oE LX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AA X 62 IRX 65 X 66 LQAYRHN (SEQ ID NO: 5);
MGSWX5EFX 8 X9RLXi 2 AIXi5Xi6RLXi9ALGGSEAELAX3oFEX33X34lAX37FEX4oX 4iLQX 44 YKGKGNPEVEALX 57 X 58 EAX 6 iAIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);
Î08\Î½Î§5Î§6Î¡ÎÎ§9Î§Î¹Î¿ÎÎÎ§Î3ÎÎÎ§ 16 Î§ 17 ÎÎÎÎÎÎ¹ÎÎÎÎÎÎ§2 8 Î¡ÎÎ§3ÎÎ§32ÎÎÎ§35Î¡ÎÎ§38 X 39 LQX 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);
MGSWAEFKQRLAAIKTRLEALZ1EAELAAFX30X31EIX34AFX37X38ELX41AYZ2N PEVEALX 52 X 53 EAX 56 AIX 59 X 6 oELX 63 AYRHN (SEQ ID NO:8);
MGSWX 5 EFX 8 X 9 RLXi 2 AIXi 5 Xi 6 RLXi 9 ALZiEAELAAF
EKEIAAFESELQAYZ 2 NPEVEX 5 oLRX 53 X 54 AAX 57 lRX 6 oX 6 iLQAYRHN (SEQ ID NO: );
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKXi 6 Xi 7 LEALZiEAELAAFX 3 oX 3 iEIX 34 AFX 37 X 38 EL X41AYZ2NPEVEX50LRX53X54AAX57IRX60X61 LQAYRHN (SEQ ID NO: 10); and
X 9 RLXi 2 AIXi 5 Xi 6 RLXi 9 ALZiEAELAX 28 FEX 3 iX 32 lAX 35 FEX 38 X 39 LQX 42 YZ 2 EVEAL X 52 X 53 EAX 56 AIX 59 X 6 oELX 63 A RHN (SEQ ID NO: 11); and combinations thereof; 

 wherein X n is a natural or non-natural amino acid; wherein each X n is the same or different natural or non-natural amino acid; and/or
The method of claim 28, wherein the polypeptide agent is attached to the solid support by covalent bonding.
The method of claim 28, wherein the polypeptide agent is attached to the solid support by non-covalent association.
A method according to any one of claims 28-30, wherein the solid support comprises a bead, glass slide, chip, gelatin, or agarose.
A composition comprising a solid support coupled to a polypeptide agent having an amino acid sequence comprising a sequence selected from the group consisting of:
FEX 33 X 3 4lAX 37 FEX4oX4iLQX44YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2);
MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 4 oELX 43 AY KGKGNPEVEALX 57 58 EAX 6 iAIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);
MGSWXsEFXiiXyRLXnAIXisXieRLXiyALGGSEAELAAFEKEIAAFESELQAYK GKGNPE VEX 55 LRX 5 8X59 AAÂ¾ 2 IRX 65 X 66 LQ AYRFIN (SEQ ID NO:4);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKX 16 X 17 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 4 oE LX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AA X 62 IRX 65 X 66 LQAYRHN (SEQ ID NO: 5);
MGSWX5EFX 8 X 9 RLXi2AIXi5Xi6RLXi9ALGGSEAELAX 3 oFEX3 3 X 3 4lAX 37 FEX 4 oX 4iLQX 44 YKGKGNPEVEALX 57 X 58 EAX 6 iAIX 64 X 65 ELX 68 AYRHN (SEQ ID NO:6);
MGSWX5X6FKX9XioLAXi3lKXi 6 Xi 7 LEALZiEAELAX 28 FEX3iX3 2 IAX35FEX 3 8 X 39 LQX 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7);
MGSWAEFKQRLAAIKTRLEALZiEAELAAFX 3 oX 3 iEIX 34 AFX 37 X 38 ELX 4 iAYZ 2 N PEVEALX52 X53EAX56AIX59X6oELX 63 AYRHN (SEQ ID NO:8); 

 MGSWXsEFXsXyRLXnAIXisXieRLXiyALZiEAELAAF
EKEIAAFESELQAYZ 2 NPEVEX 5 oLRX 53 X 54 AAX 57 lRX 6 oX 6 iLQAYRHN (SEQ ID NO: 9);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKX 16 X 17 LEALZiEAELAAFX 3 oX 3 iEIX 34 AFX 37 X 38 EL X41AYZ2NPEVEX50LRX53X54AAX57IRX60X61 LQAYRHN (SEQ ID NO: 10); and
X 9 RLXi 2 AIXi 5 Xi 6 RLXi 9 ALZ 1 EAELAX 28 FEX 3 iX 32 lAX 35 FEX 38 X 39 LQX 42 YZ 2 NPEVEAL X 52 X 53 EAX 56 AIX 59 X 60 ELX 63 AYRHN (SEQ ID NO: 11); and combinations thereof;
and wherein the amino acid sequence is not SEQ ID NO: 1
The composition of claim 32, wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: l by an amino acid substitution at one or more residues.
The composition of claim 33, wherein the amino acid substitution at one or more residues comprises a conservative substitution.
The composition of claim 33, wherein the amino acid substitution at one or more residues comprises a non-conservative substitution.
A composition according to any one of claims 33-35, wherein the amino acid substitution at one or more residues comprises a substitution at a solvent accessible residue.
The composition of any of claims 33-35, wherein the amino acid substitution at one or more residues comprises a substitution at a solvent inaccessible residue.
The composition of claim 33, wherein the polypeptide agent has an amino acid sequence that differs from SEQ ID NO: l by an amino acid deletion at one or more residues.
A composition according to any one of claims 32 to 38 wherein the solid support comprises a bead, glass slide, chip, gelatin, or agarose.
A composition comprising a polypeptide agent conjugated to a detectable agent and/or wherein the polypeptide agent has an amino acid sequence comprising a sequence selected from the group consisting of:
MGSWXsXeFKXgXioLAXoIKXigXnLEALGGSEAELAXso
MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 40 ELX 43 AY KGKGNPEVEALX 57 X 58 EAÂ¾ X 64 X 65 ELX 68 AYRHN (SEQ ID NO:3);
MGSWX5EFX8X9RLX12AIX15X16RLX19ALGGSEAELAAFEKEIAAFESELQAYK GKGNPE VEX 55 LRX 58 X 59 AAX 62 IRX 65 X 66 LQ AYRUN (SEQ ID NO:4);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKXi 6 Xi 7 LEALGGSEAELAAFX 32 X 33 EIX 36 AFX 39 X 4 oE LX 43 AYKGKGNPEVEX 55 LRX 58 X 59 AA X 62 lRX 65 X 66 LQAYRHN (SEQ ID NO: 5);
MGSWX5EFX 8 X9RLXi 2 AIXi5Xi6RLXi9ALGGSEAELAX3oFEX33X34lAX37FEX4oX 4iLQX 44 YKGKG PEVEALX57X58EAX6iAlX64X65ELX68AY H (SEQ ID NO:6);
MGSWX5X6FKX9XioLAXi3lKX 16 X 1 7LEALZiEAELAX28FEX3iX32lAX35FEX 3 8 X 39 LQX 42 YZ 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 7);
MGSWAEFKQRLAAIKTRLEALZ 1 EAELAAFX 30 X 31 EIX 34 AFX 37 X 38 ELX 41 AYZ 2 N PEVEALX 52 X 53 EAX 56 AIX 59 X 6 oELX 63 AYRHN (SEQ ID NO:8);
EKEIAAFESELQAYZ 2 NPEVEX 50 LRX 53 X 54 AAX 57 IRX 60 X 61 LQAYRHN (SEQ ID NO: 9);
MGSWX 5 X 6 FKX 9 XioLAXi 3 lKXi 6 Xi 7 LEALZiEAELAAFX 3 oX 3 iEIX 34 AFX 37 X 38 EL X4iAYZ 2 NPEVEX5oLRX53X54AAX57lPX 6 oX6i LQAYRHN (SEQ ID NO: 10); and
X 9 RLX, 2 AIX, 5X1 6 RLX, 9ALZ1 EAELAX 28 FEX31 X32IAX35FEX38X39LQX42YZ2NPEVEAL X 52 X 53 EAX 56 AIX 59 X 6 oELX 63 AYRHN (SEQ ID NO: 11); and combinations thereof;
wherein Z \ and/or Z 2 is 2 to 30 natural or non-natural amino acids;
The composition of claim 40, wherein the detectable agent comprises a chromogen.
The composition of claim 40, wherein the detectable agent comprises a fluorescent dye.
The composition of claim 40, wherein the detectable agent comprises a radionuclide.
A composition according to any one of claims 40-43, wherein the detectable agent is quantifiable.
A composition according to any one of claims 40-44, wherein the polypeptide agent is conjugated to a chromatography bead, resin, glass slide, chip, gelatin, or agarose.
The composition of claim 40, wherein the tag comprises polyhistidyl tag, a myc tag, or a FLAG tag.
The composition of claim 40, wherein the polypeptide agent is conjugated to a detectable agent or tag by covalent binding.
A composition according to any one of claims 40-47, wherein the polypeptide agent is a fusion protein.
A composition according to any one of claims 40-48, wherein the polypeptide agent is multimeric.
Ref APP-18_055096: Claims:
A D Domain target binding polypeptide that specifically binds CD123 and comprises the amino acid sequence of any one of SEQ ID NOs: 8, 13, or 14.
A polypeptide comprising the D domain of claim 1 fused to a heterologous polypeptide.
The polypeptide of claim 2, wherein the heterologous polypeptide comprises, (a) a full-length antibody or an antibody fragment; (b) an Fc domain; (c) a transmembrane domain; (d) a membrane associating domain; (e) human serum albumin or a fragment thereof; (f) AFP or a fragment thereof; (g) AFP p26 or a fragment thereof; or (h) the extracellular domain of a receptor or a fragment thereof.
(a) a full-length antibody or an antibody fragment;
(c) a transmembrane domain;
(d) a membrane associating domain;
(e) human serum albumin or a fragment thereof;
(f) AFP or a fragment thereof;
(g) AFP p26 or a fragment thereof; or
(h) the extracellular domain of a receptor or a fragment thereof.
The polypeptide of claim 2, which is labeled or conjugated to a therapeutic or cytotoxic agent.
An isolated polynucleotide encoding the polypeptide of claim 2.
A method of producing the polypeptide of claim 2, comprising culturing a host cell comprising a polynucleotide encoding the polypeptide under suitable conditions to produce the polypeptide.
A pharmaceutical composition comprising the polypeptide of claim 2 and a pharmaceutically acceptable excipient.
A chimeric antigen receptor (CAR) which comprises a target binding domain comprising the D domain that specifically binds CD123 of claim 1.
The CAR of claim 8 further comprising a transmembrane domain and an intracellular signaling domain.
The CAR of claim 9, wherein (a) the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain; (b) the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof; and/or (c) the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
(a) the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain;
(b) the intracellular signaling domain is selected from the group consisting of a domain of a human T cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof; and/or
(c) the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.
The CAR of claim 8, comprising the amino acid sequence of any one of SEQ ID NOs: 65, 66, or 67.
An isolated polynucleotide encoding the CAR of claim 8.
A lentiviral vector comprising the polynucleotide of claim 12.
A host cell comprising the polynucleotide of claim 12.
A cell engineered to express the CAR of claim 5.
The cell of claim 15, which is a T cell or a natural killer (NK) cell.
A pharmaceutical composition comprising a cell expressing the CAR of claim 8 and a pharmaceutically acceptable excipient.
The pharmaceutical composition of claim 17, wherein the cell is a T cell or a natural killer (NK) cell.
An adapter comprising (a) the D domain target binding domain that specifically binds CD123 of claim 1, and (b) an antigenic determinant (AD).
The adapter of claim 19, wherein the AD comprises an AFP p26 polypeptide.
The adapter of claim 20, wherein the AFP p26 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37-43 and 44.
The adapter of claim 10, wherein the adapter comprises two D domains that specifically bind CD123.
A method of delivering an immune response to a target cell or killing a target cell, the method comprising: (a) contacting a composition comprising the target cell with a cell expressing a chimeric antigen receptor (CAR) comprising (i) the D domain that binds to CD123 of claim 1, (ii) a transmembrane domain, and (iii) an intracellular domain; (b)
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