Patent Description:
Multiple Myeloma (MM) is the third most common haematological malignancy with <NUM>,<NUM> cases globally per year. Despite advances in treatment, MM remains one of the few haematological malignancies with an unmet medical need. Once patients progress through front-line therapy and have relapsed or refractory (r/r) disease, treatment options are very limited. However, in the recent years anti- MM tumor target antigens (TAA) have been developed. One anti-CD38 antibody, daratumumab, has been approved for the treatment of patients with relapsed MM and other anti-CD38 antibodies are currently in development (isatuximab and MOR-<NUM>, which is described in U. patent <CIT>). However, there is a need to improve responses that are currently in the range of <NUM>-<NUM>%. It was demonstrated that the activity of anti-CD38 antibodies may be enhanced by immunomodulatory therapeutics (e.g. lenalidomide), which stimulate the immune system of patients. Additionally it was demonstrated that one of the mechanisms of resistance of MM tumor cells to antibody therapies is associated with the increased signalling of checkpoint inhibitor pathways (e.g.PD-<NUM>/PD-L1). Therefore, there is an opportunity to enhance cytotoxicity of anti-CD38 antibodies against MM tumor cells and simultaneously activate the immune system by inhibiting checkpoint inhibitor pathways (e.g. PD-<NUM>/PD-L1).

In physiological conditions, PD-L1 plays a major role as guard against autoimmunity by down-regulating the immune system. It is expressed on immune "APC-like" cells (T cells, NK cells, macrophages, myeloid DCs, B cells, epithelial cells, vascular endothelial cells) and tumor cells. PD-L1 binds to its cognate receptors PD-<NUM> and B7-<NUM>, and negatively regulates immune cells (T cells, NK cells, etc), by inhibiting their proliferation and activation.

In pathological conditions, PD-L1 is highly expressed by tumor cells (> <NUM>% MM patients) and is associated with poor prognosis. Blocking antibodies targeting immune checkpoint pathways (anti-PD-<NUM>, anti-CTLA-<NUM>, anti-PD-L1, etc) have demonstrated remarkable activity in different types of cancer (lung, melanoma etc). Signs of efficacy have been observed in MM, however the activity of this class of promising therapeutics is still suboptimal in MM. One of the reasons could be that molecules, which possess beneficial activity/side effect profile (e.g. anti-PD-L1) require near stoichiometric blocking/saturation of their targets to elicit maximal immunostimulatory effect on T cells.

Therefore, specific targeting of anti-PD-L1 antibodies to the site of tumors (e.g. targeting CD38+ cancer cells) may help delivering anti-PD-L1 therapeutics to the site where immune stimulation is required, and may result in maximal immune cell stimulation allowing the complete blocking of PD-L1 on tumor and microenvironment cells. Such targeted immune cell activation at tumor sites may also reduce systemic activation of the immune cells, prevent adverse side effects, and permit higher dosing of therapeutic antibodies.

Genmab publicly announced studies of daratumumab in combination with atezolizumab in a solid tumor and multiple myeloma (announcement dated March <NUM>, <NUM>).

In a poster dated <NPL>et al reported the efficacy of a bispecific antibody anti-CD38 x anti-CD3 in treating multiple myeloma.

To harness the cytotoxic capacity of T cells, BK cells and other immune cells for the treatment of multiple myeloma (MM) and other cancers, preferably CD38+ cancers, bispecific molecules with two binding sites (specific for CD38 and PD-L1 respectively) were designed. The bispecific molecules of the invention remove the inhibition of the immune system associated with the interaction of PD-<NUM> on T cells and NK cells and PD-L1, expressed on tumor and tumor microenvironment cells. Such molecules are useful in treating cancers, especially multiple myeloma or any CD38+ cancers, which overexpress PD-L1, and grow in the microenvironments of PD-L1 expressing immune cells (Plasmacytoid Dendritic Cell, Myeloid-derived Suppressor Cells) that further inhibit T cells and NK cells. The molecules of the invention facilitate the Antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and complement-dependent cytotoxicity (CDC) of CD38+/PD-L1+ tumor cells as well as PD-L1+ cells of tumor microenvironment cells.

According to the invention, bispecific CD38/PD-L1 antibodies, comprising anti-CD38 and anti-PD-L1 domains, are engineered. These bispecific CD38/PD-L1 antibodies are capable of simultaneous binding to both antigens.

In a preferred embodiment, bispecific CD38/PD-L1 antibodies are expressed in CHO cells and are purified by affinity chromatography employing Protein A resins. Antibody binding properties are characterized in in vitro assays. They simultaneously bind both CD38 and PD-L1 in ELISA assay.

The bispecific tetravalent four Fab antibodies, having the structure of <FIG>B are designated BiXAb®, a trademark of Biomunex Therapeutics.

The antibody of the invention is a bispecific and bivalent for CD38 and PD-L1. The antigen-binding bispecific antibodies of the invention are full-length bispecific antibodies consisting of a continuous "composite heavy chain" (made of the natural heavy chain of IgG of mAb1 followed by Linkers and the Fab heavy chain of mAb2), which is constructed of an Fc (Hinge-CH2-CH3) followed by antibody <NUM> Fab heavy chain (CH1-VH) and the successive Fab heavy chain (CH1-VH) of antibody <NUM>, the latter joined by a hinge-derived polypeptide linker sequence, and the resulting composite heavy chain during protein expression, associates with the identical second composite heavy chain, while the co-expressed Fab light chains (LC) of antibody <NUM> and of antibody <NUM> associate with their cognate heavy chain domains in order to form the final tandem F(ab')<NUM>-Fc molecule; the antibody <NUM> (Ab1) and the antibody <NUM> (Ab2) being different and selected from the group consisting of an anti-CD38 antibody (daratumumab) or its mutated derivatives and an anti-PD-L1 antibody (atezolizumab) or its mutated derivatives.

The BiXAb® antibodies are able to bind bivalently both to CD38 and PD-L1.

Still another object of the invention is a method for preparing the bispecific antibodies of the invention, said method comprising the following steps: a) culturing in suitable medium and culture conditions a host cell expressing an antibody heavy chain as defined above, and antibody light chains as defined above; and b) recovering said produced antibodies from the culture medium or from said cultured cells.

The invention makes use of recombinant vectors, in particular expression vectors, comprising polynucleotides encoding the heavy and light chains defined herein, associated with transcription- and translation-controlling elements which are active in the host cell chosen. Vectors which can be used to construct expression vectors in accordance with the invention are known in themselves, and will be chosen in particular as a function of the host cell one intends to use. Preferably, said host cell is transformed with a polynucleotide encoding a heavy chain and two polynucleotides encoding two different light chains. Said polynucleotides can be inserted in a same expression vector, or in separate expression vectors. The method for producing the antibodies of the invention comprises culturing such host-cell and recovering said antigen-binding fragments or antibody from said culture.

The basic structure of a naturally occurring antibody molecule is a Y-shaped tetrameric quaternary structure consisting of two identical heavy chains and two identical light chains, held together by non-covalent interactions and by inter-chain disulfide bonds.

In mammalian species, there are five types of heavy chains: α, δ, ε, γ, and µ, which determine the class (isotype) of immunoglobulin: IgA, IgD, IgE, IgG, and IgM, respectively. The heavy chain N-terminal variable domain (VH) is followed by a constant region, containing three domains (numbered CH1, CH2, and CH3 from the N-terminus to the C-terminus) in heavy chains γ, α, and δ, while the constant region of heavy chains µ and ε is composed of four domains (numbered CH1, CH2, CH3 and CH4 from the N-terminus to the C-terminus). The CH1 and CH2 domains of IgA, IgG, and IgD are separated by a flexible hinge, which varies in length between the different classes and in the case of IgA and IgG, between the different subtypes: IgG1, IgG2, IgG3, and IgG4 have respectively hinges of <NUM>, <NUM>, <NUM> (or <NUM>), and <NUM> amino acids, and IgA1 and IgA2 have respectively hinges of <NUM> and <NUM> amino acids.

There are two types of light chains: λ and <IMG>, which can associate with any of the heavy chains isotypes, but are both of the same type in a given antibody molecule. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a constant region consisting of a single domain termed CL.

The heavy and light chains pair by protein/protein interactions between the CH1 and CL domains, and via VH /VL interactions and the two heavy chains associate by protein/protein interactions between their CH3 domains. The structure of the immunoglobulin molecule is generally stabilized by interchains disulfide bonds between the CH1 and CL domains and between the hinges.

The antigen-binding regions correspond to the arms of the Y-shaped structure, which consist each of the complete light chain paired with the VH and CH1 domains of the heavy chain, and are called the Fab fragments (for Fragment antigen binding). Fab fragments were first generated from native immunoglobulin molecules by papain digestion which cleaves the antibody molecule in the hinge region, on the amino-terminal side of the interchains disulfide bonds, thus releasing two identical antigen-binding arms. Other proteases such as pepsin, also cleave the antibody molecule in the hinge region, but on the carboxy-terminal side of the interchains disulfide bonds, releasing fragments consisting of two identical Fab fragments and remaining linked through disulfide bonds; reduction of disulfide bonds in the F(ab')<NUM> fragments generates Fab' fragments.

The part of the antigen binding region corresponding to the VH and VL domains is called the Fv fragment (for Fragment variable); it contains the CDRs (complementarity determining regions), which form the antigen-binding site (also termed paratope).

The effector region of the antibody which is responsible of its binding to effector molecules or cells, corresponds to the stem of the Y-shaped structure, and contains the paired CH2 and CH3 domains of the heavy chain (or the CH2, CH3 and CH4 domains, depending on the class of antibody), and is called the Fc (for Fragment crystallisable) region.

Due to the identity of the two heavy chains and the two light chains, naturally occurring antibody molecules have two identical antigen-binding sites and thus bind simultaneously to two identical epitopes.

An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being assessed for treatment and/or being treated. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, rabbit, dog, etc..

The term "treatment" or "treating" refers to an action, application or therapy, wherein a subject, including a human being, is subjected to medical aid with the purpose of improving the subject's condition, directly or indirectly. Particularly, the term refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combination thereof in some embodiments. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. For example, with respect to cancer, "treatment" or "treating" may refer to slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof.

The invention provides bispecific tetravalent antibodies, comprising two binding sites to each of their targets, and a functional Fc domain allowing the activation of effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and complement-dependent cytotoxicity (CDC).

The antibodies of the invention are full-length antibodies. They preferably comprise heavy chains and light chains from human immunoglobulins, preferably IgG, still preferably IgG1.

The light chains preferably are Kappa light chains.

In a preferred embodiment, the linker of the invention connects two pairs of IgG Fab domains in a tetra-Fab bispecific antibody format, the amino acid sequence of which comprises the heavy chain sequences of at least two Fab joined by a linker, followed by the native hinge sequence, followed by the IgG Fc sequence, coexpressed with the appropriate IgG light chain sequences.

An example of the antibodies of the invention, which have an IgG-like structure, is illustrated in <FIG>.

The bispecific antibodies of the invention comprise.

the antibody <NUM> (Ab1) and the antibody <NUM> (Ab2) being different.

Ab1 and Ab2, being different, independently are selected from the group consisting of an anti-CD38 antibody (daratumumab) and an anti-PD-L1 antibody (atezolizumab).

Daratumumab binds a unique CD38 epitope at the C-terminal region of human CD38, amino acids <NUM> to <NUM> and <NUM> to <NUM>, with amino acids in positions <NUM> and <NUM> being particularly important for binding. Advantageously, Ab1 and/or Ab2 may be antibodies that bind to the same epitope, or overlapping epitope (e.g. with an overlap of at least <NUM> amino acids) with respect to daratumumab.

It is herein disclosed a bispecific antibody which comprises, preferably consists of, a) two heavy chains, each comprising, preferably consisting of, SEQ ID NO:<NUM> and b) four light chains, two comprising, preferably consisting of, SEQ ID NO:<NUM>, the two others comprising, preferably consisting of, SEQ ID NO: <NUM>. Such bispecific antibody is designated BiXAb-<NUM>.

In a preferred embodiment, the bispecific molecule is a bispecific antibody which comprises, preferably consists of, a) two heavy chains, each comprising, preferably consisting of, SEQ ID NO:<NUM> and b) four light chains, two comprising, preferably consisting of, SEQ ID NO:<NUM>, the two others comprising, preferably consisting of, SEQ ID NO: <NUM>. Such bispecific antibody is designated BiXAb-<NUM>.

The polypeptide linker, also designated "hinge-derived polypeptide linker sequence" or "pseudo hinge linker", comprises all or part of the sequence of the hinge region of one or more immunoglobulin(s) selected among IgA, IgG, and IgD, preferably of human origin. Said polypeptide linker may comprise all or part of the sequence of the hinge region of only one immunoglobulin. In this case, said immunoglobulin may belong to the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain is derived, or to a different isotype or subclass. Alternatively, said polypeptide linker may comprise all or part of the sequences of hinge regions of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal portion of the polypeptide linker, which directly follows the CH1 domain, preferably consists of all or part of the hinge region of an immunoglobulin belonging to the same isotype and subclass as the immunoglobulin from which said CH1 domain is derived.

Optionally, said polypeptide linker may further comprise a sequence of from <NUM> to <NUM>, preferably of from <NUM> to <NUM> N-terminal amino acids of the CH2 domain of an immunoglobulin.

The polypeptide linker sequence typically consists of less than <NUM> amino acids, preferably less than <NUM> amino acids, still preferably less than <NUM> amino acids.

In some cases, sequences from native hinge regions can be used; in other cases point mutations can be brought to these sequences, in particular the replacement of one or more cysteine residues in native IgG1, IgG2 or IgG3 hinge sequences by alanine or serine, in order to avoid unwanted intra-chain or inter-chains disulfide bonds.

In a particular embodiment, the polypeptide linker sequence comprises or consists of amino acid sequence EPKX<NUM>CDKX<NUM>HX<NUM>X<NUM>PPX<NUM>PAPELLGGPX<NUM>X7PPX<NUM>PX<NUM>PX<NUM>GG (SEQ ID NO:<NUM>), wherein X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, identical or different, are any amino acid. In particular, the polypeptide linker sequence may comprise or consist of a sequence selected from the group consisting of.

A non-limitative example of a hinge-derived polypeptide linker which can be used in a multispecific antigens-binding fragment of the invention is a polypeptide having SEQ ID NO:. Said polypeptide consists of the full length sequence of human IgG1 hinge, followed by the <NUM> N-terminal amino-acids of human IgG1 CH2 (APELLGGPS, SEQ ID NO: <NUM>), by a portion of the sequence of human IgA1 hinge (TPPTPSPS, SEQ ID NO: <NUM>), and by the dipeptide GG, added to provide supplemental flexibility to the linker. In another preferred embodiment, the hinge-derived polypeptide linker sequence is SEQ ID NO: <NUM> or SEQ ID NO:<NUM>.

In a particular embodiment, X<NUM>, X<NUM> and X<NUM>, identical or different, are Threonine (T) or Serine (S).

In another particular embodiment, X<NUM>, X<NUM> and X<NUM>, identical or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I), still preferably X<NUM>, X<NUM> and X<NUM>, identical or different, may be Ala (A) or Gly (G).

Alternatively, X<NUM>, X<NUM> and X<NUM>, identical or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

In a particular embodiment, X<NUM> and X<NUM>, identical or different, are any amino acid selected from the group consisting of Serine (S), Cysteine (C), Alanine (A), and Glycine (G).

In a preferred embodiment, X<NUM> is Serine (S) or Cysteine (C).

In a preferred aspect, X<NUM> is Alanine (A) or Cysteine (C).

In a particular embodiment, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, identical or different, are any amino acid other than Threonine (T) or Serine (S). Preferably X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, identical or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I).

Alternatively, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, identical or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

In a preferred embodiment, X<NUM>, X<NUM>, X<NUM>, X<NUM>, X<NUM>, identical or different, are selected from the group consisting of Ala (A) and Gly (G).

In still a preferred embodiment, X<NUM> and X<NUM> are identical and are preferably selected from the group consisting of Ala (A) and Gly (G).

In a preferred embodiment, the polypeptide linker sequence comprises or consists of sequence SEQ ID NO: <NUM>, wherein.

In another preferred embodiment, the polypeptide linker sequence comprises or consists of sequence SEQ ID NO: <NUM>, wherein.

The skilled person may refer to <CIT>, for general techniques of expressing multispecific antibodies.

Also herein described is a polynucleotide comprising a sequence encoding a protein chain of the antibody of the invention. Said polynucleotide may also comprise additional sequences: in particular it may advantageously comprise a sequence encoding a leader sequence or signal peptide allowing secretion of said protein chain. Host-cells transformed with said polynucleotide are also disclosed.

Typically, the amino acid sequences of different anti-CD38 and anti-PDL-<NUM> monoclonal antibodies are used to design the DNA sequences, optionally after codon optimization for mammalian expression. For the heavy chain, the DNAs encoding signal peptides, variable region and constant CH1 domain of Fab1 followed the hinge linker and variable region and constant CH1 domain of Fab2 with flanking sequences for restriction enzyme digestion are synthesized. For the light chain, the DNAs encoding signal peptides and variable and constant Kappa regions are synthesized.

Nucleic acids encoding heavy and light chains of the antibodies of the invention are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences.

In one example, both the heavy and light chain-coding sequences (e.g., sequences encoding a VH and a VL, a VH-CH1 and a VL-CL, or a full-length heavy chain and a full-length light chain) are included in one expression vector. In another example, each of the heavy and light chains of the antibody is cloned into an individual vector. In the latter case, the expression vectors encoding the heavy and light chains can be co-transfected into one host cell for expression of both chains, which can be assembled to form intact antibodies either in vivo or in vitro. Alternatively, the expression vector encoding the heavy chain and that or those encoding the light chains can be introduced into different host cells for expression each of the heavy and light chains, which can then be purified and assembled to form intact antibodies in vitro.

In a particular embodiment, a host cell is co-transfected with three independent expression vectors, such as plasmids, leading to the coproduction of all three chains (namely the heavy chain HC, and two light chains LC1 and LC2, respectively) and to the secretion of the bispecific antibody.

More especially the three vectors may be advantageously used in a following molecular ratio of <NUM>:<NUM>:<NUM> (HC : LC1 : LC2).

The recombinant vectors for expression the antibodies described herein typically contain a nucleic acid encoding the antibody amino acid sequences operably linked to a promoter, either constitutive or inducible. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the antibody. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

Bispecific antibodies as described herein may be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, plant, insect (e.g. using a baculovirus vector), and mammalian cells. It is not necessary that the recombinant antibodies of the invention are glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are human embryonic kidney line (<NUM> cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells/- or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells).

Mammalian tissue cell culture is preferred to express and produce the polypeptides because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines (such as NS0), or transformed B-cells or hybridomas.

In a most preferred embodiment, the bispecific antibodies of the invention are produced by using a CHO cell line, most advantageously CHO-S or CHO-DG-<NUM> cell lines or their derivatives.

Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, or cytomegalovirus.

The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally <NPL>). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.

Host cells are transformed or transfected with the vectors (for example, by chemical transfection or electroporation methods) and cultured in conventional nutrient media (or modified as appropriate) for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

The expression of the antibodies may be transient or stable.

Preferably, the bispecific antibodies are produced by the methods of stable expression, in which cell lines stably transfected with the DNA encoding all polypeptide chains of a bispecific antibody, such as BiXAb-<NUM>, are capable of sustained expression, which enables manufacturing of therapeutics. For instance stable expression in a CHO cell line is particularly advantageous.

Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be further isolated or purified to obtain preparations that substantially homogeneous for further assays and applications. Standard protein purification methods known in the art can be used. For example, suitable purification procedures may include fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, high-performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, and gel filtration (see generally <NPL>). Substantially pure immunoglobulins of at least about <NUM> to <NUM>% homogeneity are preferred, and <NUM> to <NUM>% or more homogeneity most preferred, for pharmaceutical uses.

In vitro production allows scale-up to give large amounts of the desired bispecific antibodies of the invention. Such methods may employ homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges.

The polypeptide sequences that bind CD38 derive from daratumumab or its mutated derivatives.

The polypeptide sequences that bind PD-L1 derive from atezolizumab or its mutated derivatives.

The term "mutated derivative", "mutant", or "functional variant" designates a sequence that differs from the parent sequence to which it refers by deletion, substitution or insertion of one or several amino acids. Preferably the mutated derivative preferably show at least <NUM>%, preferably at least <NUM>%, still preferably at least <NUM>% homology sequence with the native sequence. In a particular embodiment, the mutations do not substantially impact the function of the antibody.

Mutated derivatives, or functional variants, can comprise a VH chain that comprises an amino acid sequence at least <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%) identical to any of the reference sequences recited herein, a VL chain that has an amino acid sequence at least <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%) identical to any of the reference sequences recited herein, or both. These variants are capable of binding to CD38 and PD-L1. In some examples, the variants possess similar antigen-binding affinity relative to the reference antibodies described above (e.g., having a KD less than <NUM> × <NUM>-<NUM> M, <NUM>-<NUM> M, preferably less than <NUM> × <NUM>-<NUM> or <NUM> × <NUM>-<NUM> M).

The affinity of the binding is defined by the terms ka (associate rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). Typically, specifically binding when used with respect to an antibody refers to an antibody that specifically binds to ("recognizes") its target(s) with an affinity (KD) value less than <NUM>-<NUM> M, preferably less than <NUM>-<NUM> M, e.g., less than <NUM>-<NUM> M or <NUM>-<NUM> M. A lower KD value represents a higher binding affinity (i.e., stronger binding) so that a KD value of <NUM>-<NUM> indicates a higher binding affinity than a KD value of <NUM>-<NUM>.

The "percent identity" of two amino acid sequences is determined using the algorithm of <NPL>, modified as in <NPL>. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version <NUM>) of <NPL>. BLAST protein searches can be performed with the XBLAST program, score=<NUM>, wordlength=<NUM> to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in<NPL>. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In other embodiments, the functional variants described herein can contain one or more mutations (e.g., conservative substitutions) which preferably do not occur at residues which are predicted to interact with one or more of the CDRs.

It is herein described mutated derivatives, or functional variants, which are substantially identical to the reference antibody.

The term "substantially identical" or "insubstantial" means that the relevant amino acid sequences (e.g., in framework regions (FRs), CDRs, VH, or VL domain) of a variant differ insubstantially (e.g., including conservative amino acid substitutions) as compared with a reference antibody such that the variant has substantially similar binding activities (e.g., affinity, specificity, or both) and bioactivities relative to the reference antibody. Such a variant may include minor amino acid changes, e.g. <NUM> or <NUM> substitutions in a <NUM> amino acid sequence of a specified region. Generally, more substitutions can be made in FR regions, in contrast to CDR regions, as long as they do not adversely impact the binding function of the antibody (such as reducing the binding affinity by more than <NUM>% as compared to the original antibody). In some embodiment, the sequence identity can be about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or higher, between the original and the modified antibody. In some embodiments, the modified antibody has the same binding specificity and has at least <NUM>% of the affinity of the original antibody.

Conservative substitutions will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with another residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art. For example, amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein. Variants comprising one or more conservative amino acid substitutions can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. <NPL>, or <NPL>. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

The present disclosure also provides antibody variants with improved biological properties of the antibody, such as higher or lower binding affinity, or with altered ADCC properties on CD38 and/or PD-L1 expressing cells.

Amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or via peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to achieve the final construct, provided that the final construct possesses the desired characteristics. Nucleic acid molecules encoding amino acid sequence variants of the antibody can be prepared by a variety of methods known in the art.

These methods include oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant (natural) version of the antibody. In one embodiment, the equilibrium dissociation constant (KD) value of the antibodies of the invention is less than <NUM>-<NUM> M, particularly less than <NUM>-<NUM> M, <NUM>-<NUM> M or <NUM>-<NUM> M. The binding affinity may be determined using techniques known in the art, such as ELISA or biospecific interaction analysis (e.g. using surface plasmon resonance), or other techniques known in the art.

Any of the molecules described herein can be examined to determine their properties, such as antigen-binding activity, antigen-binding specificity, and biological functions, following routine methods.

Any of the molecules described herein can be modified to contain additional nonproteinaceous moieties that are known in the art and readily available, e.g., by PEGylation, hyperglycosylation. Modifications that can enhance serum half-life are of interest.

Throughout the present description, amino acid sequences are defined according to <NPL>).

Mutations can be located in constant domains. The bispecific antibodies indeed advantageously comprise Fab fragments having mutations at the interface of the CH1 and CL domains, said mutations facilitate cognate pairing of heavy chain/light chain and preventing their mispairing.

In a preferred embodiment, bispecific antibodies are described herein, which comprise.

the Fab fragments being tandemly arranged in the following order.

In particular examples, bispecific antibodies are described, wherein the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain that derives from the CH1 domain of an immunoglobulin by substitution of the threonine residue at position <NUM> of said CH1 domain with an aspartic acid and the cognate CL domain is a mutated domain that derives from the CL domain of an immunoglobulin by substitution of the asparagine residue at position <NUM> of said CL domain with a lysine residue and substitution of the serine residue at position <NUM> of said CL domain with an alanine residue, and/or wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutated domain that derives from the CH1 domain of an immunoglobulin by substitution of the leucine residue at position <NUM> of said CH1 domain with a glutamine and substitution of the serine residue at position <NUM> of said CH1 domain with a valine residue, and the cognate CL domain is a mutated domain that derives from the CL domain of an immunoglobulin by substitution of the valine residue at position <NUM> of said CL domain with a threonine residue and substitution of the serine residue at position <NUM> of said CL domain with a valine residue.

The antibodies of the invention may be glycosylated or not, or may show a variety of glycosylation profiles. In a preferred embodiment, antibodies are unglycosylated on the variable region of the heavy chains, but are glycosylated on the Fc region.

Certain mutated derivatives may use humanized forms of the reference antibody. In a humanization approach, complementarity determining regions (CDRs) and certain other amino acids from donor mouse variable regions are grafted into human variable acceptor regions and then joined to human constant regions. See, e.g.<NPL>); <CIT>.

The bispecific antibody of the invention is useful as a medicament, in particular in treating a cancer.

The term "cancer" as used herein includes any cancer, especially a hematological malignancy, and any other cancer characterized by CD38 or PD-L1 expression or overexpression, and especially those cancers characterized by co-expression of both CD38 and PD-L1.

Examples of cancers are lymphoma or leukemia, such as Non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), or multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma.

The bispecific molecule as defined herein may formulated in a composition, e.g. a pharmaceutical composition, containing a bispecific molecule as defined herein, formulated together with a pharmaceutical carrier.

As used herein, a "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, or isotonic and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

The composition can be administered by a variety of methods known in the art. The route and/or mode of administration will vary depending upon the desired results.

To administer the bispecific antibody of the invention by certain routes of administration, it may be necessary to coat the bispecific antibody of the invention with, or co-administer the bispecific antibody of the invention with a material to prevent its inactivation. For example, the bispecific antibody of the invention may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sodium chloride into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, which delay absorption.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. For example the bispecific molecule or antibody of the invention can be administrated at a dosage of <NUM>-<NUM>/kg from <NUM> times/week to <NUM> time/month.

The present invention, thus generally described above, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting the instant invention.

The amino acid sequences of different anti-CD38 and anti-PDL-<NUM> monoclonal antibodies were used to design the DNA sequences after codon optimization for mammalian expression using GeneScript program. For the heavy chain, the DNAs encoding signal peptides, variable region and constant CH1 domain of Fab1 followed the hinge linker and variable region and constant CH1 domain of Fab2 with flanking sequences for restriction enzyme digestion were synthesized by GeneScript. For the light chain, the DNAs encoding signal peptides and variable and constant Kappa regions were synthesized by GeneScript.

PCR reactions using PfuTurbo Hot Start were carried out to amplify the inserts which were then digested by Notl + Apal and Notl + Hindlll for heavy and light chains, respectively. The double digested heavy chain fragments were ligated with Notl + Apal digested Evitria's proprietary expression vector in which the human IgG1 CH1 + hinge + CH2 + CH3 domains were already inserted. The double digested light chain fragments were ligated with Notl + Hindlll treated Evitria's proprietary vector. Plasmid DNAs were verified by double strand DNA sequencing.

For a <NUM> scale expression, a total of <NUM>µg of plasmid DNAs in Evitria's proprietary vector (<NUM>µg heavy chain + <NUM>µg of each light chain, LC1 and LC2) were mixed in <NUM> Eppendorf tube, <NUM> of CHO SFM medium containing <NUM>µL of <NUM>/mL PEI pH7. <NUM> was added, incubated at RT for <NUM>. The mixture of DNA-PEI was loaded into <NUM> of FreeStyle™ CHO-S cells at <NUM>-<NUM> × <NUM><NUM> cells/mL in <NUM> shaking flask. Cells were shaken for <NUM> more days. The supernatant was harvested by centrifuging cells at <NUM>,<NUM> rpm for <NUM>.

The harvested supernatant was purified by Protein A resin. Electrophoresis was performed under reducing conditions and non-reducing conditions employing Gel Biorad Stain-Free <NUM> - <NUM>% gels and the corresponding running buffer. Samples were prepared by combining the purified BiXAb® antibodies with 2X SDS sample buffer and heating for <NUM> at <NUM>. Preparation of reduced samples included the addition of NuPAGE reducing agent prior to heating. The apparent MW was determined using Ladder Precision Plus Protein Unstained Standards (Biorad). <FIG> presents the SDS-PAGE pattern of CD38/PD-L1 antibodies under reducing conditions. Two bands corresponding to the composite heavy chain and two co-migrating light chains are observed and are of the expected molecular weight. <FIG> presents the SDS-PAGE pattern of CD38/PD-L1 antibodies under non-reducing conditions.

The dominant band at <NUM> kDa corresponds to the complete CD38/PD-L1 BiXAb® molecule as expected.

For Dual Antigen Binding Plate ELISA Assay the following reagents were used: Recombinant human CD38, Fc-tagged (Creative BioMart); biotinylated Human PD-L1, Avi Tag (AcroBiosystems); Streptavidin-HRP, (Biotechne RD-Systems). Human CD38-Fc fusion protein was coated with <NUM>µL/well at <NUM>µg/mL in 1X PBS pH7. <NUM> in Maxisorp plates at <NUM> overnight. The plates were washed <NUM> times with 1X PBS containing <NUM>% Tween-<NUM> (1X PBST), then blocked with <NUM>% non-fat milk/1X PBST at <NUM>µL/well with shaking at RT for <NUM> hr. <NUM>µL/well of BiXAb® <NUM> and BiXAb® <NUM> at <NUM>/ ml stock solution starting at <NUM>/<NUM> dilution in 1X PBS at <NUM>:<NUM> series dilutions were added. The plates were incubated at RT for <NUM> hr with shaking, followed by <NUM> washes with 1X PBST. <NUM>µL/well of <NUM>µg/mL Biotin-human PD-L1 protein in 1X PBS was added and plates were shaken at RT for <NUM> hr. After <NUM> washes with 1X PBST, <NUM>µL/well of <NUM>. 1µg/mL of Streptavidin-conjugated HRP in 1X PBS was added. The plates were shaken at RT for <NUM> hr followed by <NUM> washes with 1X PBST. <NUM>µL/well TMB substrate in 1X PBS was added for color development. The data were collected at <NUM> for <NUM> sec per well on a Victor II multifunction plate reader. <FIG> demonstrates the dual antigen binding profiles of two CD38/PD-L1 BiXAbs®. This profile confirms that both types of binding domains of these molecules (anti-CD38 domains and anti-PD-L1 domains) bind their cognate antigen targets.

The amino acid sequences of anti-CD38 (daratumumab) and anti-PDL1 (atezolizumab) were used to design the DNA sequences, after codon optimization for mammalian expression, using the GeneScript program. These antibodies are referred to as the "parental" anti-CD38 and the "parental" anti-PD-L1 mAbs.

The DNA construct of the heavy chain was designed as such: signal peptide (SEQ ID NO:<NUM>), followed by sequence SEQ ID NO:<NUM> [consisting of the variable region, followed by the constant CH1 domain of Fab1 (anti-CD38), in which mutations Leu to Gln and Ser to Val at Kabat positions <NUM> and <NUM> were introduced, respectively, followed by the linker, followed by the variable region, followed by the constant CH1 domain of Fab2 (anti-PD-L1), in which mutation Thr to Asp at Kabat position <NUM> was introduced]; flanking sequences for restriction enzyme digestion were introduced on both ends of the heavy chain DNA construct. The DNA construct for the light chain was designed as such: signal peptide (SEQ ID NO:<NUM>), followed by the variable region, followed by the constant Kappa region. For the anti-CD38 light chain, mutations where introduced at Kabat positions <NUM> (Leu to Gln) and <NUM> (Ser to Val) in the constant Kappa domain. For the anti-PDL1 light chain, mutations at Kabat positions <NUM> (Val to Thr) and <NUM> (Ser to Val) were introduced into the constant Kappa domain. All DNA constructs were synthesized by Gene Art.

PCR reactions, using PfuTurbo Hot Start, were carried out to amplify the inserts, which were then digested with Notl and Apal, and Notl and Hindlll for heavy and light chains, respectively. The double digested heavy chain fragments were ligated with Notl and Apal treated pcDNA3. <NUM> expression vector (Invitrogen) into which the human IgG1 hinge followed by the CH2-CH3 domains were already inserted. The double-digested light chain fragments were ligated with Notl and Hindlll treated pcDNA3. <NUM> expression vector (Invitrogen). Plasmid DNAs were verified by double strand DNA sequencing.

The bispecific antibody BiXAb-<NUM> was produced employing transient gene expression by co-transfecting <NUM> genes coded on separate vectors in a <NUM>:<NUM>:<NUM> = HC:LC1:LC2 molecular ratio (<NUM> continuous heavy chain (HC) and <NUM> light chains (LC)) in CHO-S cells adapted to serum-free medium in suspension (CHO SFM-II medium, Life Technologies™). Typically, for <NUM> scale expression, a total of <NUM>µg of plasmid DNA (<NUM>µg heavy chain, <NUM>µg of anti-CD38 light chain and <NUM>µg of anti-PD-L1 light chain) were mixed in a <NUM> Eppendorf tube, then <NUM> of CHO SFM medium containing <NUM>µL of <NUM>/mL PEI transfection reagent pH7. <NUM> (Polyplus) was added, and the reaction incubated at room temperature for <NUM>. The DNA-PEI mixture was subsequently added to <NUM> of Life Technologies' Invitrogen FreeStyle™ CHO-S cells at <NUM>~2x <NUM>/mL in a <NUM> shake flask. Cells were shaken for <NUM> days. The supernatant was harvested by centrifugation at <NUM>,<NUM> rpm for <NUM>. The expression titer of BiXAb-<NUM> in the supernatant was determined using ForteBio's protein A biosensors (Octet® Systems). BiXAb-<NUM> was then purified on protein A affinity resin (MabSelect SuRe, GE Healthcare Life Sciences). The antibody was eluted from protein A using <NUM> glycine pH <NUM>, and the eluate was neutralized by <NUM> TRIS. The purified antibody, in Dulbecco's PBS (Lonza), was sterile-filtered (<NUM> sterile filters, Techno Plastic Products AG), and the final concentration determined by reading the optical density (OD) at <NUM> (Eppendorf BioSpectrometer®).

BiXAab-<NUM> typically exhibited good expression titer (> <NUM> / liter) in transient CHO expression. This level of expression is comparable to the level of expression seen with conventional monoclonal antibodies.

In order to evaluate the quality of purified BiXAb-<NUM>, we performed SDS-PAGE (Experion™ automated electrophoresis system, BioRad). In the presence of sodium dodecyl sulfate (SDS) in the running buffer, the rate at which an antibody migrates in the gel depends primarily on its size, enabling molecular weight determination. This assay was performed under non-reducing conditions and under reducing conditions; the latter permits disruption of the disulfide bonds, and hence visualization of individual polypeptide chains (the light chains and the heavy chain).

The SDS-PAGE data are presented in <FIG>. Under non-reducing conditions, the quaternary structure of the antibody is maintained, and the molecular weight observed should represent the sum of the molecular weights of the different heavy and light chains.

The bispecific antibody of the invention (BiXAb-<NUM>) consists of six chains: two heavy chains and four light chains. The theoretical molecular weight of BiXab-<NUM> is <NUM> kDa, not accounting for post-translational modifications (PTM), e.g. N-glycosylation in the Fc at asparagine <NUM>. The gel was calibrated using a mixture of standards of known molecular weight. The non-reducing data exhibit a major band running close to the <NUM> kDa molecular weight standard, which is in accordance with the calculated molecular weight and the expected glycosylation of two asparagines at position <NUM> in the Fc domain. Under reducing conditions, dithiothreitol (DTT) further denatures BiXAb-<NUM> by reducing the disulfide linkages and breaking the quaternary structure, and thus the six polypeptide chains should migrate separately in the gel according to their molecular weight. The two identical heavy chains of BiXAb-<NUM> co-migrate as a single band, and the two pairs of light chains, due to their nearly identical molecular weight, co-migrated as the second band. Therefore, the data exhibit two major bands, at approximately <NUM> kDa and <NUM> kDa, based on the mobility of the molecular weight standards. Each heavy chain possessed one N-glycosylation site at asparagine <NUM>, which explains the broadness of the higher molecular weight band and the observed molecular weight slightly higher than calculated (<NUM> kDa); this broadening is typical for glycosylated proteins. The calculated molecular weights of the light chains of anti-CD38 (<NUM> kDa) and anti-PD-L1 (<NUM> kDa) are very similar, and thus resulted in their co-migration.

In conclusion, the SDS-PAGE of BiXAb-<NUM> exhibited the expected profiles, under both non-reducing and reducing conditions, and was in agreement with the calculated theoretical molecular weights, when accounting for the existence of an N-glycosylation site in the heavy chain.

Protein aggregation is frequently observed in engineered protein molecules. We performed analytical size exclusion chromatography (SEC) to assay the high molecular weight species content of the single-step affinity-purified BiXAb-<NUM> preparation (see Expression and Purification of variants). We employed an SEC-s3000 (<NUM>× <NUM>) column (BioSep) and an Aktapurifier <NUM> system (GE Healthcare); the assay was conducted at a flow rate of <NUM>/min using PBS buffer pH <NUM>.

The SEC chromatogram presented in <FIG> demonstrated that the main peak corresponded to the expected size of the monomeric BiXAb-<NUM>; this peak represented <NUM>% of the total sample. In addition, a small peak corresponding to higher molecular weight species (possibly dimers) was observed; this peak represented <NUM>% of the total sample. Thus, we concluded that the percentage content of higher molecular weight species is minor, and is similar to conventional monoclonal antibodies produced in CHO expression systems. The narrow and symmetric shape of the monomeric peak suggested that BiXAb-<NUM> was correctly assembled and was represented by a single species.

Differential Scanning Calorimetry (DSC) was used to compare the thermal stability of BiXAb-<NUM>, the parental anti-CD38 mAb, and the parental anti-PD-L1 mAb. A MicrocalTM VP-Capillary DSC system (Malvern Instruments) was used to perform differential scanning calorimetry experiments.

All samples were centrifuged (<NUM>,000x g, <NUM>, <NUM>), and their protein content was quantitated prior to the DSC analysis using a Nanodrop ND-<NUM> spectrophotometer (Thermo Scientific) employing the IgG analysis program. For assay, all samples were diluted in PBS to a final concentration of <NUM>/mL.

The pre-equilibration time was <NUM>, and the resulting thermograms were acquired between <NUM> and <NUM> at a scan rate of <NUM>/h, a filtering period of <NUM> sec, and medium feedback. Prior to sample analysis, <NUM> buffer/buffer scans were measured to stabilize the instrument, and a buffer/buffer scan was performed between each protein/buffer scan. The data were fit to a non-<NUM>-state unfolding model, with the pre- and post- transition adjusted by subtraction of the baseline.

The DSC curves presented in <FIG> (covering the <NUM> to <NUM> range) demonstrated the manner in which individual Fv regions can lead to different Fab unfolding profiles; this experiment also demonstrated that the Fv regions dictate the apparent stabilities of the Fabs. The DSC profile of the anti-CD38 mAb exhibited two transitions: a large peak having a Cp max of <NUM> Kcal/mole/oC and a Tm1 of <NUM> oC, corresponding to the unfolding of both CH2 and Fab domains, and a small peak having a Cp max of <NUM> Kcal/mole/oC and a Tm2 of <NUM>. 5oC, corresponding to the unfolding of the CH3 domain. The DSC profile of the anti-PD-L1 mAb exhibited two transitions: a small peak having a Cp max of <NUM> Kcal/mole/oC and a Tm1 of <NUM> oC, corresponding to the unfolding of the CH2 domain, and a large peak having a Cp max of <NUM> Kcal/mole/oC and a Tm2 of <NUM> oC, corresponding to the unfolding of both CH3 and Fab domains.

The DSC profile of BiXAb-<NUM> also exhibited two transitions with two large peaks. The first peak had a Cp max of <NUM> Kcal/mole/oC and a Tm1 of <NUM> oC, and corresponded to the unfolding of the CH2 and Fab domains of the anti-CD38 mAb; the second peak had a Cp max of <NUM> Kcal/mole/oC and a Tm2 of <NUM> oC, and corresponded to the unfolding of the CH3 and Fab domains of the anti-PD-L1 mAb. Thus, the DSC profile of BiXAb-<NUM> resembled the superposition of the two DSC profiles of the two parental mAbs, and illustrated the excellent assembly and stability of BiXAb-<NUM>. The Tonset of BiXAb-<NUM> (<NUM> oC) was similar to that of the parental mAbs (anti-CD38 Tonset=<NUM> oC and anti-PD-L1 Tonset=<NUM> oC), indicating that BiXAb-<NUM> possessed stability properties similar to those of the parental antibodies. The calculated ΔH of BiXAb-<NUM> was <NUM> kcal/mole, reflecting the larger size of the bispecific molecule relative to the two parental antibodies (anti-CD38 ΔH=<NUM> kcal/mole and anti-PD-L1 ΔH=<NUM> kcal/mole).

<NUM>µl of either parental mAb, anti-CD38 or anti-PDL1, each at a concentration of <NUM>µg/mL, prepared by dilution with PBS pH <NUM>, were used to coat Maxisorp plates at <NUM> overnight. Also, BiXAb-<NUM>, at a concentration of <NUM>µg/mL, prepared by dilution with PBS pH <NUM>, was used to coat Maxisorp plates at <NUM> overnight. The plates were washed <NUM> times with 1x PBS containing <NUM>% Tween-<NUM> (PBST), and then blocked with <NUM>µL/well <NUM>% BSA in 1x PBS at room temperature for <NUM> hrs. The plates were subsequently washed <NUM> times with 1x PBST. A seven-point <NUM>-fold dilution series of recombinant CD38 His/Flag-tagged (Creative Biomart) in 1x PBS, starting at <NUM>µg/mL, was prepared; <NUM>µL of each dilution step was added per assay well. The plates were incubated at room temperature for <NUM> hr, and washed <NUM> times with 1x PBST. <NUM>µL/well of anti-Flag-tag antibody-conjugated HRP (Abcam), diluted <NUM>,<NUM>-fold in 1x PBS, was added and the plates were incubated at room temperature for <NUM> hr. After <NUM> washes with 1x PBST, <NUM>µL/well of TMB substrate in 1x PBS was added for colorimetric readout, and the plates incubated for <NUM> at room temperature for color development. The assay data were collected employing a Victor2 microplate reader (Perkin Elmer) at <NUM>.

BiXAb-<NUM> exhibited a dose-dependent binding curve very similar to that of the parental anti-CD38 antibody (<FIG>). The EC50 of CD38 binding for both antibodies were as follows: EC50[BiXAb-<NUM>] = <NUM> ng/mL and EC50[anti-CD38] = <NUM> ng/mL. This result suggested that BiXAb-<NUM> possessed correctly assembled anti-CD38 Fab domains, since it exhibited binding similar to that of the parental anti-CD38 mAb. The parental anti-PDL1 mAb, used as a negative control, did not exhibit any binding, as expected.

<NUM>µL of biotinylated human PD-L1 protein (AcroBiosystems) at a concentration of <NUM>µg/mL, prepared by dilution with 1x PBS pH7. <NUM>, was used to coat Maxisorp plates at <NUM> overnight. The plates were washed <NUM> times with PBST, and then blocked with <NUM>µL/well <NUM>% BSA in 1x PBS at room temperature for <NUM> hrs. The plates were subsequently washed <NUM> times with 1x PBST. Seven-point <NUM>-fold dilution series of either the anti-CD38 mAb (starting at <NUM>/mL), or the anti-PD-L1 mAb (starting at <NUM>/mL), or BiXAb-<NUM> (starting at <NUM>/mL) in 1x PBS were prepared; <NUM>µL of each dilution step was added per assay well. The plates were incubated at room temperature for <NUM> hr and washed <NUM> times with 1x PBST. <NUM>µL/well of anti-human antibody (IgG H&L)-conjugated HRP (Abliance), diluted <NUM>,<NUM>-fold in 1x PBS, was added, and the plates were incubated at room temperature for <NUM> hr. After <NUM> washes with 1x PBST, <NUM>µL/well of TMB substrate in 1x PBS was added for colorimetric readout, and the plates incubated for <NUM> at room temperature for color development. The assay data were collected employing a Victor2 microplate reader (Perkin Elmer) at <NUM>.

BiXAb-<NUM> exhibited a dose-dependent binding curve very similar to that of the parental anti-PD-L1 antibody (<FIG>). The EC50 of PD-L1 binding for both antibodies were as follows: EC50[BiXAb-<NUM>] = <NUM> ng/mL and EC50[anti-PD-L1] = <NUM> ng/mL. This result suggested that BiXAb-<NUM> possessed correctly assembled anti-PD-L1 Fab domains, since it exhibited binding similar to that of the parental anti-PD-L1 mAb. The parental anti-CD38 mAb, used as a negative control, did not exhibit any binding, as expected.

<NUM>µL of recombinant human Fc-tagged CD38 (Creative BioMart), at <NUM>µg/mL prepared by dilution with 1x PBS pH7. <NUM>, was used to coat Maxisorp plates at <NUM> overnight. The plates were washed <NUM> times with 1x PBST, and then blocked with <NUM>µL/well <NUM>% BSA in 1x PBS at room temperature for <NUM> hrs. The plates were washed <NUM> times with 1x PBST. A seven-point three-fold dilution series in 1x PBS of BiXAb-<NUM> (starting at <NUM>µg/mL) was prepared, and <NUM>µL of each dilution step was added per assay well. The plates were incubated at room temperature for <NUM> hr, and subsequently washed <NUM> times with 1x PBST. <NUM>µL/well of <NUM>µg/mL biotinylated human PD-L1 (AcroBiosystems) in 1x PBS was added, and the plates were incubated at room temperature for <NUM> hr. After <NUM> washes with 1x PBST, <NUM>µL/well of <NUM>µg/mL of streptavidin-conjugated HRP (Biotechne) prepared by dilution with 1x PBS was added. The plates were incubated at room temperature for <NUM> hr. After <NUM> washes with 1x PBST, <NUM>µL/well of TMB substrate in 1x PBS was added for colorimetric readout, and the plates incubated for <NUM> at room temperature for color development. The assay data were collected employing a Victor2 microplate reader (Perkin Elmer) at <NUM>.

BiXAb-<NUM> exhibited a dose-dependent binding curve in the dual ELISA format, suggesting that it possessed correctly assembled anti-CD38 and anti-PD-L1 Fab domains (<FIG>). This demonstrated that BiXAb-<NUM> is a bispecific antibody capable of binding CD38 and PD-L1 simultaneously with EC50 = <NUM> ng/ mL. Neither of the two parental mAbs, anti-CD38 or anti-PDL1, exhibited any binding in this dual ELISA format, as expected.

CHO-CD38 cells (CHO cells stably transfected with full length human CD38) were cultured in DMEM-Glutamax-I medium supplemented with <NUM>µg/ml penicillin, <NUM>µg/ml streptomycin, <NUM>% fetal calf serum and <NUM>µg/ml geneticin. SKOV-<NUM> cells and RPMI-<NUM> cells were cultured in RPMI <NUM>-Glutamax-I medium, supplemented with <NUM>µg/ml penicillin, <NUM>µg/ml streptomycin, and <NUM>% fetal calf serum.

3x105 cells (CHO-CD38, or SKOV-<NUM>, or RPMI-<NUM>) per each sample were used. Cells were washed 1x with the PBA solution (PBS supplemented with <NUM>%BSA and <NUM>% Na-azide). For the determination of the FACS profiles, the cells were stained with the respective antibodies at a concentration of 50µg/ml in a volume of <NUM>µl. For the titration of BiXAb-<NUM> and the parental anti-CD38 antibody, and subsequent determination of the binding parameters, CHO-CD38 cells were stained with the respective antibodies at the indicated concentrations in a volume of <NUM>µl. Cells were incubated for <NUM> on ice and then washed <NUM> times with <NUM> of PBA solution. Cells were incubated with fluorescently labelled anti-human kappa or anti-human IgG Fc gamma specific secondary antibodies on ice in the dark for <NUM>, and then washed <NUM> times with <NUM> PBA solution; lastly, cells were re-suspended in a final volume of <NUM>µl PBA solution. Samples were assayed using either an Epics-XL or a Navios flow cytometer (Beckman Coulter). <NUM> events were acquired in each experiment.

The binding profiles of BiXAb-<NUM> and the parental anti-CD38 and anti-PD-L1 parental antibodies are presented in <FIG>We chose to test a multiple myeloma cell line, RPMI-<NUM>, which expresses high levels of CD38 and negligible levels of PD-L1 (<FIG>); a CHO-CD38 cell line that expressed a very high level of CD38 due to stably transfected full length CD38 (<FIG>); and an ovarian cancer cell line SKOV-<NUM>, which is known to express PD-L1 (<FIG>). These profiles exhibited a single peak for BiXAb-<NUM> that was very similar to the profiles of both parental antibodies on the <NUM> cell lines. This suggested that BiXAb-<NUM> is correctly folded and possesses binding attributes similar to those of the parental antibodies. As expected, CHO-CD38 expressed only CD38 and no PD-L1, whereas SKOV-<NUM> expressed only PD-L1 and no CD38.

In order to quantitatively confirm that the binding properties of BiXAb-<NUM> are similar to those of the parental anti-CD38 antibody, a titration of BiXAb-<NUM> and the anti-CD38 parental antibody was performed employing CHO-CD38 cells, as presented in <FIG>. The EC50 of BiXAb-<NUM> was determined to be <NUM> and that of the parental anti-CD38 was <NUM>, confirming the similar binding properties of the anti-CD38 Fab domains in BiXAb-<NUM> and in the parental anti-CD38 antibody. Negative controls in this experiment, anti-PD-L1 and anti-CD20 antibodies, demonstrated no binding to CHO-CD38 cells, as expected.

CHO-CD38, SKOV-<NUM>, and RPMI-<NUM> cells were cultured as described in Example <NUM> above. For preparation of MNC the following procedure was employed. Freshly drawn peripheral blood was anti-coagulated with citrate. Subsequently, <NUM> of Ficoll-Paque PLUS solution was layered with <NUM> anti-coagulated whole blood. Samples were centrifuged for <NUM> at <NUM>,<NUM> rpm at RT with no subsequent centrifuge breaking. MNC were collected from the plasma / Ficoll interface. The MNC cell suspension was diluted <NUM>:<NUM> in PBS and centrifuged for <NUM> minutes at <NUM>,<NUM> rpm at room temperature. The supernatant was removed, and the erythrocytes were lysed by addition of <NUM> ice-cold distilled water to the cell suspension for <NUM> seconds, after which <NUM> of 10x PBS was added. The cells were centrifuged for <NUM> at <NUM> rpm at room temperature and washed with 1x PBS three times to remove platelets. Finally cells were re-suspend in <NUM> cell culture medium. Cell numbers were adjusted to achieve <NUM>:<NUM>= Effector cell : Tumor cell ratio in the ADCC assays.

For the ADCC <NUM>Chromium release assay, <NUM>×<NUM><NUM> target cells (RPMI <NUM>, SKOV-<NUM>, or CHO-CD38) were incubated with 100µCi 51Chromium in <NUM>µl PBS for <NUM> hours at <NUM> and <NUM>% CO2. After <NUM> hours incubation, cells were washed three times with <NUM> of medium and finally re-suspended at a concentration of <NUM> × <NUM> cells/ml. Target cells (<NUM>,<NUM> cells/well) and MNC in the presence of antibodies were incubated in a <NUM>-well micro-titer plate (200µl assay volume) for <NUM> hours at <NUM> and <NUM>% CO2. For the determination of maximal target cell lysis (= maximal cpm) Triton X-<NUM> was added. To determine basal <NUM>Chromium release (= basal cpm) target cells were not further manipulated. After 4hr incubation, micro-titer plates were centrifuged for <NUM> at <NUM> rpm and <NUM>µl supernatant was mixed with <NUM>µl of Optiphase Supermix (Perkin Elmer) and incubated in a shake incubator for <NUM>. Samples were assayed in a MicroBeta TriLux (Perkin Elmer) beta-counter instrument. Target cell lysis was calculated using the following formula:<MAT>.

All of the measurements were performed in triplicate.

ADCC assays of CD38+ cells (RPMI-<NUM> and CHO-CD38) were performed employing non-pre-activated MNC as effector cells (<FIG>) The assays showed potent cytotoxicity of BiXAb-<NUM> and the anti-CD38 antibody on RPMI-<NUM> cells with EC50 of <NUM> and <NUM>, respectively; on CHO-CD38 cells, the cytotoxicity of BiXAb-<NUM> and the anti-CD38 antibody had EC50 of <NUM> and <NUM>, respectively. Anti-PD-L1 showed minimal activity on both cells lines; two negative control mAbs, anti-CD20 and anti-HER2, did not facilitate any lysis, as expected. These results demonstrate the potent ADCC activity of BMX-<NUM> against CD38+ cells, which is similar to that of the parental anti-CD38 antibody.

SKOV3 cells, RPMI <NUM>, and CHO-CD38 cells were cultured as described in Example <NUM>. MNC were prepared as described in Example <NUM>. NK cells were isolated from MNC by negative selection employing the "NK cell isolation kit, human" (Miltenyi) according to the manufacturer's instructions. NK cells were cultivated over night at a seeding density of <NUM>×<NUM><NUM> cells / ml in RPMI medium supplemented with <NUM>% fetal calf serum. IL-<NUM> or IL-<NUM> was added to a final concentration of <NUM> ng/ml. ADCC assays were performed as outlined in Example <NUM> with the exception that the Effector cell : Tumor cell ratio was kept at <NUM>:<NUM> and the duration of the reaction was reduced to <NUM> hr.

Claim 1:
A bispecific molecule comprising at least one anti-CD38 domain and at least one anti-PD-L1 domain, which are capable of simultaneous binding to CD38 and PD-L1 antigens, respectively, which bispecific molecule is a full length antibody comprising two heavy chains and four light chains,
wherein each heavy chain comprises
d. a Fc region comprising Hinge-CH2-CH3 domains,
e. which Fc region is linked to Fab heavy chain (CH1-VH) of antibody <NUM> (Ab1),
f. which in turn is linked to the Fab heavy chain (CH1-VH) of antibody <NUM> (Ab2), by a hinge-derived polypeptide linker sequence, wherein said polypeptide linker sequence links the N-terminus of said Fab heavy chain VH domain of Ab1 with the C-terminus of said CH1 domain of Ab2,
and the four light chains comprise Fab light chains (CL-VL) of Ab1 and Fab light chains (CL-VL) of Ab2 associated with their cognate heavy chain domains;
wherein Ab1 is atezolizumab and Ab2 is daratumumab.