Stable multispecific antibodies

Abstract

The present invention relates to multispecific antibody constructs comprising Fab fragments having a specific set of mutations at the interface of the CH1 and CL domains, which mutations prevent heavy chain / light chain mismatching.

Description

[0001] The present invention relates to the production of multispecific antibody molecules useful in the medical field. Background of the Invention

[0003] Bispecific antibodies (bsAbs) combine the specificities of two antibodies and target different antigens or epitopes simultaneously. BsAbs with a "two-target" function can interfere with various surface receptors or ligands associated with, for example, cancer, proliferation, or inflammatory processes. BsAbs can also bring targets into close proximity to support the formation of protein complexes on one cell or to trigger cell-to-cell contact. Examples of the "forced-linkage" function are bsAbs that support protein complexation in the coagulation cascade or bsAbs that target tumor-immunocyte recruiters and / or activators. After years of research and development, the first bsAb was approved in 2009.

[0004] Initially, bispecific antibodies were prepared by chemical conjugation or by using quadromas generated by fusing two hybridoma cell lines that produce two different mAbs. However, chemical conjugation may sometimes alter the antigen-binding site, resulting in impaired biological properties of the antibody. The quadroma method has the drawback that the random pairing of heavy and light chains from two different antibodies theoretically generates ten equally possible combinations, resulting in a mixture of immunoglobulin molecules, only one of which is the desired bispecific product and must be separated from the mispaired products.

[0005] Genetic engineering has now become the preferred method for producing bispecific antibodies and has led to the development of a variety of different recombinant bispecific antibody formats. Some of these bispecific antibodies are very simple and are derived from single-chain Fv (scFv) fragments of two (or more) different antibodies linked by an appropriate peptide linker. These antibodies are relatively easy to produce, and since they are formed by a single polypeptide chain and contain only the Fv regions of the parental antibodies, there is no problem of mismatching between the chains. However, they are smaller than full-length immunoglobulins and do not have constant regions, especially the Fc region. Although this may be advantageous in some applications, such as when it is desired to avoid Fc-mediated effects, it is disadvantageous when Fc-mediated effector functions are required. Moreover, due to their small size and lack of an Fc region, they have a very short in vivo half-life.

[0006] Therefore, other bispecific recombinant antibody formats have been designed that more closely mimic naturally occurring immunoglobulin molecules, especially those with a complete Fc region. They can be divided into two main formats.

[0007] In the first type of antibody (IgG scFv), the scFv fragment from antibody A is fused to the end (usually the C-terminus) of the heavy chain of antibody B. The resulting antibody has only one type of heavy chain (which contains the VH, CH1, CH2, and CH3 domains of antibody B and the VH and VL domains of antibody A), and one type of light chain (which contains the VL and CL domains of antibody B), and there is no mismatch between the chains. For example, Qu et al. (Blood, 111, 2211-2219, 2008) described this form.

[0008] In the second type of antibody, the heavy and light chains of antibody A are paired with the heavy and light chains of antibody B. This form regenerates the bispecific antibodies produced by quadromas and thus also has a similar problem of mismatch. To solve the problem of heavy chain mismatching pairs, it has been proposed to mutate the CH3 domain of the antibody to promote its heterodimerization (i.e., heavy chain A pairs with heavy chain B) and prevent its homodimerization. This is accomplished by the so-called "knob into holes" method (Ridgway et al., Protein Eng, 9, 617-21, 1996; US Patent 7,695,936). A "knob" mutation is introduced at the CH3 dimer interface of the heavy chain of antibody A, which consists of replacing a smaller amino acid with a larger amino acid, creating steric hindrance that prevents homodimerization. At the same time, to promote heterodimerization, a complementary "hole" mutation consisting of replacing a large amino acid with a smaller amino acid is introduced into the CH3 domain of antibody B. To solve the problem of heavy chain / light chain mismatch, it has been proposed to use antibodies with different specificities but sharing a common light chain, which has been previously identified from an scFv phage library (Merchant et al., Nat Biotechnol, 16, 677-81, 1998; US Patent 7,183,076). The disadvantage of this method is that it is difficult to identify antibodies with a common light chain. International Patent Application WO2013 / 005194 proposes that heavy chain / light chain mismatch can be prevented by mutating some key residues at the interface of the CH1 and CL domains, and thus ensuring the desired pairing of the chains. Summary of the Invention

[0010] The inventors have now discovered that a new set of mutations can further improve the chain matching in multispecific antibody molecules.

[0011] The present invention relates to multispecific, e.g., bispecific antibody constructs, which comprise different Fab fragments having a specific set of mutations at the interface of the CH1 and CL domains, said mutations promoting homologous pairing of the heavy chain / light chain and preventing their mismatch.

[0012] The sequence position numbers used herein for the CH1 and CL domains refer to the Kabat numbering (Kabat, E. A. et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH publication, No. 91 - 3242, pp. 662, 680, 689, 1991).

[0013] This disclosure provides mutant Fab fragments selected from the following:

[0014] a) A Fab fragment comprising:

[0015] - the VH and VL domains of the antibody of interest;

[0016] - a CH1 domain derived from the CH1 domain of an immunoglobulin by substituting the threonine residue at position 192 of the CH1 domain with an aspartic acid residue, and

[0017] - a CL domain derived from the CL domain of an immunoglobulin by substituting the asparagine residue at position 137 of the CL domain with a lysine residue and substituting the serine residue at position 114 of the CL domain with an alanine residue; and

[0018] b) A Fab fragment comprising:

[0019] - the VH and VL domains of the antibody of interest;

[0020] - a CH1 domain derived from the CH1 domain of an immunoglobulin by substituting the leucine residue at position 124 of the CH1 domain with a glutamine residue and substituting the serine residue at position 188 of the CH1 domain with a valine residue; and

[0021] - a CL domain derived from the CL domain of an immunoglobulin by substituting the valine residue at position 133 of the CL domain with a threonine residue and substituting the serine residue at position 176 of the CL domain with a valine residue.

[0022] Any construct comprising such a mutant Fab fragment, preferably any protein construct, is part of the present invention.

[0023] In particular, multispecific antigen - binding fragments are provided, which comprise at least two Fab fragments having different CH1 and CL domains, wherein each Fab fragment recognizes a different epitope of interest, and the Fab fragments are arranged in tandem in any order, with the C - terminus of the CH1 domain of the first Fab fragment being linked to the N - terminus of the VH domain of the subsequent Fab fragment via a polypeptide linker,

[0024] At least one Fab fragment, more preferably two fragments selected from:

[0025] a) A Fab fragment consisting of:

[0026] A CH1 domain derived from the CH1 domain of an immunoglobulin by substituting the threonine residue at position 192 of the CH1 domain with an aspartic acid residue, and

[0027] - A CL domain derived from the CL domain of an immunoglobulin by substituting the asparagine residue at position 137 of the CL domain with a lysine residue and substituting the serine residue at position 114 of the CL domain with an alanine residue; and

[0028] b) A Fab fragment consisting of:

[0029] - The VH and VL domains of the antibody of interest;

[0030] - A CH1 domain derived from the CH1 domain of an immunoglobulin by substituting the leucine residue at position 124 of the CH1 domain with a glutamine residue and substituting the serine residue at position 188 of the CH1 domain with a valine residue; and

[0031] - A CL domain derived from the CL domain of an immunoglobulin by substituting the valine residue at position 133 of the CL domain with a threonine residue and substituting the serine residue at position 176 of the CL domain with a valine residue.

[0032] According to a preferred embodiment, the CH1 domain is derived from an IgG immunoglobulin, advantageously the IgG1 subtype. The CL domain is preferably of the κ type. Preferably, when used for human therapy, the immunoglobulin from which the mutated CH1 and mutated CL domains are derived is a human immunoglobulin.

[0033] The VH and VL domains can be derived from any natural or genetically engineered antibody that recognizes the epitope one wishes to target.

[0034] The mutated Fab fragments of the present invention can be used in any multispecific antibody construct where promotion of homologous pairing of the heavy chain / light chain and prevention of their mismatching are required.

[0035] Advantageously, the mutated Fab fragments can be used in multispecific antibody molecules comprising antigen-binding fragments, each of which consists essentially of Fab fragments arranged in tandem separated by a linker.

[0036] Accordingly, another object of the present invention is a multispecific antigen-binding fragment comprising at least two and up to six different Fab fragments selected from:

[0037] - A Fab fragment containing the wild-type CH1 and CL domains of immunoglobulin (also defined herein as: "wild-type Fab fragment")

[0038] - The mutant Fab fragment (a) as defined above;

[0039] - The mutant Fab fragment (b) as defined above;

[0040] Each Fab fragment recognizes a different epitope of interest, and the Fab fragments are arranged in tandem in any order, with the C-terminus of the CH1 domain of the first Fab fragment linked to the N-terminus of the VH domain of the subsequent Fab fragment via a polypeptide linker;

[0041] Or one or two Fab fragments are arranged in tandem in any order and linked via a polypeptide linker to the C-terminus of the CH3 domain or the C-terminus of the hinge sequence, while the other Fab or two tandemly arranged Fabs are attached to the N-terminus of the hinge sequence in any order.

[0042] The above-mentioned hinge sequence is usually a natural hinge sequence obtained from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD, and most preferably obtained from IgG1, or can be a sequence modified by point mutations and / or addition of amino groups, such that the modified hinge sequence consists of less than 80 amino acids, preferably less than 60 amino acids, still more preferably less than 40 amino acids.

[0043] "Antigen-binding fragment" is defined herein as a molecule having two or more antigen-binding regions, each region recognizing a different epitope. The different epitopes can be carried by the same antigen molecule or different antigen molecules.

[0044] In a preferred embodiment, a multispecific antibody having two identical antigen-binding arms is further provided, each arm consisting of a multispecific antigen-binding fragment, i.e., an antigen-binding fragment capable of binding multiple (i.e., more than one) antigens, as described above.

[0045] In a specific embodiment, the multispecific antibody has an immunoglobulin-like structure and comprises

[0046] - Two identical antigen-binding arms, each arm consisting of such a multispecific antigen-binding fragment as defined above;

[0047] - The dimerized CH2 and CH3 domains of immunoglobulin;

[0048] - The hinge region of IgA, IgG or IgD, linking the C-terminus of the CH1 domain of the antigen-binding arm to the N-terminus of the CH2 domain.

[0049] More particularly, a subject of the invention is a multispecific, preferably bispecific antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises

[0050] - an Fc region of an immunoglobulin comprising a hinge-CH2-CH3 domain,

[0051] - said Fc region is linked via said hinge domain to the Fab heavy chain CH1-VH of antibody 1 (Ab1),

[0052] - which in turn is linked via a polypeptide linker sequence to the Fab heavy chain CH1-VH of antibody 2 (Ab2), and said polypeptide linker sequence links the N-terminus of the VH domain of the Fab heavy chain of said Ab1 to the C-terminus of the CH1 domain of said Ab2,

[0053] and the four light chains comprise the Fab light chain CL-VL of Ab1 and the Fab light chain CL-VL of Ab2 associated with their cognate heavy chain domains;

[0054] wherein Ab1 and Ab2 recognize different epitopes,

[0055] and wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutant domain which is derived from the CH1 domain of an immunoglobulin by substitution of the threonine residue at position 192 of the CH1 domain with an aspartic acid residue and the cognate CL domain is a mutant domain which is derived from the CL domain of an immunoglobulin by substitution of the asparagine residue at position 137 of the CL domain with a lysine residue and the serine residue at position 114 of the CL domain with an alanine residue, and / or

[0056] wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutant domain which is derived from the CH1 domain of an immunoglobulin by substitution of the leucine residue at position 124 of the CH1 domain with a glutamine residue and the serine residue at position 188 of the CH1 domain with a valine residue, and the cognate CL domain is a mutant domain which is derived from the CL domain of an immunoglobulin by substitution of the valine residue at position 133 of the CL domain with a threonine residue and the serine residue at position 176 of the CL domain with a valine residue.

[0057] Any protein chain selected from the following is further described:

[0058] - the light chain of the mutant Fab fragment of the invention;

[0059] - the heavy chain of the mutant Fab fragment of the invention;

[0060] - The heavy chain of the antigen-binding fragment of the present invention;

[0061] - The heavy chain of the immunoglobulin-like multispecific antibody of the present invention.

[0062] The present disclosure further provides a polynucleotide comprising a sequence encoding the protein chain of the present invention. The polynucleotide may further comprise additional sequences: in particular, it may advantageously comprise a sequence encoding a leader sequence or signal peptide that allows secretion of the protein chain. Brief Description of the Drawings

[0064] Figure 1 Shows SDS polyacrylamide gel electrophoresis of BiXAb-6567 under reducing and non-reducing conditions. Lane 1: Migration of BiXAb-6567 under reducing conditions; Lane 2: Molecular weight marker indicating the weight of each band; Lane 3: Migration of BiXAb-6567 under non-reducing conditions.

[0065] Figure 2 Shows size exclusion chromatography analysis of BiXAb-6567.

[0066] Figure 3 Shows the melting curves of two parental antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 determined by differential scanning calorimetry.

[0067] Figure 4A Shows the binding curves of two parental antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 in direct CD38 antigen-binding ELISA. Figure 4B Shows the binding curves of two parental antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 in direct PD-L1 antigen-binding ELISA. Figure 4C Shows the binding curve of BiXAb-6567 in dual antigen (PD-L1 and CD38) binding ELISA.

[0068] Figures 5A to 5C Shows fluorescence-activated cell sorting curves of two parental mAbs (anti-CD38 and anti-PD-L1) and BiXAb-6567 on three different cell lines, Figure 5A : Multiple myeloma RPMI-8226, 5B: CHO cells stably transfected with full-length CD38, and 5C: Ovarian cancer cell line SKOV-3.

[0069] Figure 6 Shows the titration binding curves of two parental antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and a negative control anti-CD20 antibody on the CHO-CD38 cell line.

[0070] Figure 7 Shows the cytotoxic activity curves of two parental antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and two negative control antibodies anti-CD20 and anti-HER2, in an ADCC assay using the multiple myeloma cell line RPMI-8226 as the target cell and ungraded non-preactivated monocytes as the effector cells.

[0071] Figure 8 Shows the cytotoxic activity curves of two parental antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and two negative control antibodies anti-CD20 and anti-HER2, in an ADCC assay using the CHO-CD38 cell line as the target cell and ungraded non-preactivated monocytes as the effector cells.

[0072] Figure 9 Shows the cytotoxic activity curves of two parental antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and two negative control antibodies anti-CD20 and anti-HER2, in an ADCC assay using the SKOV-3 cell line as the target cell and enriched IL-12-preactivated NK cells as the effector cells.

[0073] Figure 10 Shows the cytotoxic activity curves of two parental antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and two negative control antibodies anti-CD20 and anti-HER2, in an ADCC assay using the SKOV-3 cell line as the target cell and enriched IL-15-preactivated NK cells as the effector cells.

[0074] Figure 11 Is a schematic diagram of the bispecific antibody of the present invention, which comprises two heavy chains and four light chains, and shows different combinations of mutations. Detailed Description of the Invention

[0076] Definition:

[0077] The basic structure of a naturally occurring antibody molecule is a Y-shaped quaternary structure composed of two identical heavy chains and two identical light chains, which are bound together by non-covalent interactions and interchain disulfide bonds.

[0078] In mammalian species, there are five types of heavy chains: α, δ, ε, γ, and μ, which determine the class (isotype) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, respectively. After the N-terminal variable domain (VH) of the heavy chain is the constant region, which contains 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 regions of heavy chains μ and ε consist 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, the length of which varies between different types and is different between different subtypes for IgA and IgG: IgG1, IgG2, IgG3, and IgG4 have hinges of 15, 12, 62 (or 77), and 12 amino acids, respectively, while IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

[0079] There are two types of light chains: λ and κ, which can bind to any heavy chain isotype, but they are both of the same type in a given antibody molecule. The two light chains appear to be functionally identical. After their N-terminal variable domain (VL) is the constant region, which consists of a single domain called CL.

[0080] Heavy and light chains pair through protein / protein interactions between the CH1 and CL domains and through VH / VL interactions, and the two heavy chains bind through protein / protein interactions between their CH3 domains. The structure of the immunoglobulin molecule is usually stabilized by interchain disulfide bonds between the CH1 and CL domains and between the hinges.

[0081] The antigen-binding region corresponds to the arms of the Y-shaped structure and consists of each complete light chain paired with the VH and CH1 domains of the heavy chain and is called the Fab fragment (for fragment antigen-binding). The Fab fragment is first produced by digestion of the native immunoglobulin molecule with papain, which cuts the antibody molecule in the hinge region on the amino-terminal side of the interchain disulfide bond, thus releasing two identical antigen-binding arms. Other proteases (such as pepsin) can also cut the antibody molecule in the hinge region on the carboxyl-terminal side of the interchain disulfide bond, releasing a fragment consisting of two identical Fab fragments and held together by a disulfide bond; reduction of the disulfide bond in the F(ab′)2 fragment produces the Fab′ fragment.

[0082] The part of the antigen-binding region corresponding to the VH and VL domains is called the Fv fragment (variable fragment); it contains CDRs (complementary determining regions), which form the antigen-binding site (also called the paratope).

[0083] The effector region of an antibody responsible for binding to effector molecules or cells corresponds to the stem of the Y-shaped structure and contains paired CH2 and CH3 domains (or CH2, CH3, and CH4 domains, depending on the class of the antibody) of the heavy chains, and is referred to as the Fc (fragment crystallizable) region.

[0084] Due to the identity of the two heavy chains and two light chains, a naturally occurring antibody molecule has two identical antigen-binding sites and thus binds to two identical epitopes simultaneously.

[0085] In the context of the present invention, a "multispecific antigen-binding fragment" is defined herein as a molecule having two or more antigen-binding regions, each region recognizing a different epitope. The different epitopes can be carried by the same antigen molecule or different antigen molecules. The terms "recognizing" or "recognizes" mean that the fragment specifically binds to the target antigen.

[0086] A "multispecific antibody" contains at least two multispecific antigen-binding fragments / arms and is capable of binding two, three, or more different antigens.

[0087] An antibody "specifically binds" to a target antigen if it binds to the target antigen with a higher affinity, avidity, more rapidly, and / or for a longer duration compared to binding 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.

[0088] The terms "mutated derivative", "mutant", or "functional variant" denote a sequence that differs from the parental sequence it refers to by the deletion, substitution, or insertion of one or several amino acids. Preferably, the mutated derivative preferably shows a homologous sequence of at least 80%, preferably at least 85%, more preferably at least 90% with the native sequence. In certain embodiments, the mutation substantially does not affect the function of the antibody.

[0089] The mutated derivative or functional variant can comprise a VH chain (which comprises an amino acid sequence having at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%)) identity with any of the reference sequences described herein), a VL chain (which has an amino acid sequence having at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%)) identity with any of the reference sequences described herein, or both. These variants are capable of binding to the target antigen. In some instances, the variant has a similar antigen-binding affinity relative to the above-mentioned reference antibody (e.g., having less than 1x10 -7 M, 10 -8 M, preferably less than 1x10-9 or 1 x 10 -10 KD of M).

[0090] The affinity of binding is defined by the terms ka (association rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). Generally, when used with respect to an antibody, specific binding means binding with an affinity (KD) value of less than 10 -7 M, preferably less than 10 -8 M, such as less than 10 -9 M or 10 -10 M that specifically binds (``recognizes'') its target. A lower KD value indicates a higher binding affinity (i.e., stronger binding), and thus a KD value of 10 -9 represents a higher binding affinity than a KD value of 10 -8 .

[0091] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XNBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecule of interest. In cases where there are gaps between two sequences, gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0092] 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 predicted to interact with one or more CDRs.

[0093] Mutant derivatives or functional variants that are substantially identical to a reference antibody are described herein.

[0094] The terms "substantially the same" or "insubstantial" mean that the relevant amino acid sequence of the variant (e.g., in the framework region (FR), CDR, VH or VL domain) is not substantially different from the reference antibody (e.g., including conservative amino acid substitutions), such that the variant has substantially similar binding activity (e.g., affinity, specificity or both) and biological activity relative to the reference antibody. Such variants can include minor amino acid changes, such as 1 or 2 substitutions in a 5-amino acid sequence in a specified region. Generally, more substitutions can be made in the FR region compared to the CDR region, provided that they do not adversely affect the binding function of the antibody (e.g., reduce the binding affinity by more than 50% compared to the original antibody). In some embodiments, the sequence identity between the original antibody and the modified antibody can be about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, the modified antibody has the same binding specificity and has at least 50% of the affinity of the original antibody.

[0095] Conservative substitutions will result in molecules having similar functions and chemical properties to those from which such modifications are made. For example, "conservative amino acid substitutions" can involve replacing 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 in a molecular sequence or to increase or decrease the affinity of the molecules described herein. Variants containing one or more conservative amino acid substitutions can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those methods can be found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between 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.

[0096] Amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics. Nucleic acid molecules encoding amino acid sequence variants of an antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of earlier prepared variants or non-variant (native) versions of the antibody. In one embodiment, the equilibrium dissociation constant (KD) value of the antibody of the present invention is less than 10 -7 M, particularly less than 10 -8 M, 10 -9 M or 10 -10 M. Binding affinity can 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.

[0097] Any molecule described herein can be examined by conventional methods to determine its properties, such as antigen-binding activity, antigen-binding specificity, and biological function.

[0098] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. The subject can be a human, but also includes other mammals, particularly those mammals that can be used as laboratory models for human diseases, such as mice, rats, rabbits, dogs, etc.

[0099] The term "treatment" or "treating" refers to an operation, application, or therapeutic method in which a subject, including a human, is provided with medical assistance with the aim of directly or indirectly improving the subject's condition. In particular, in some embodiments, the term refers to reducing morbidity or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing morbidity, improving symptoms, improving prognosis, or a combination thereof. Those skilled in the art will understand that treatment does not necessarily result in the complete absence or elimination of symptoms. For example, with respect to cancer, "treatment" or "treating" can refer to slowing the growth, proliferation, or metastasis of a tumor or malignant cells, preventing or delaying the development of the growth, proliferation, or metastasis of a tumor or malignant cells, or some combination thereof.

[0100] Preferred design of multispecific antibodies:

[0101] Provided herein are multispecific antigen-binding fragments and multispecific antibody constructs comprising the said fragments, wherein each multispecific antigen-binding fragment consists essentially of Fab fragments separated by the linker of the present invention in tandem arrangement.

[0102] Such fragments and constructs preferably comprise chains from human immunoglobulins, preferably IgG, still more preferably IgG1.

[0103] In the case of a multispecific antigen-binding fragment comprising more than two different Fab fragments, the polypeptide linkers separating the Fab fragments can be the same or different. In a preferred embodiment of the multispecific antibody according to the invention, it has two identical antigen-binding arms, each arm consisting of an antigen-binding fragment capable of binding multiple (more than one) antigens, as defined above. Depending on the intended use of the antibody, the antigen-binding arms can be linked together in a variety of ways.

[0104] If an antibody without Fc-mediated effects is desired, the antibody will not contain an Fc region. In this case, the two antigen-binding arms can be linked together, for example:

[0105] - by homodimerization of the antigen-binding arms via interchain disulfide bonds provided by the polypeptide linker separating the Fab fragments, if the linker contains cysteine residues; and / or

[0106] - by adding a polypeptide extension containing a cysteine residue to the C-terminus of each antigen-binding arm, allowing the formation of interchain disulfide bonds and homodimerization of the polypeptide extension, resulting in a hinge-like structure; by way of non-limiting example, the polypeptide extension can be, for example, the hinge sequence of IgG1, IgG2, IgG3, IgA or IgD;

[0107] - by a semi-rigid linker that connects the C-termini of the heavy chains of the two antigen-binding arms to form a single polypeptide chain and keeps the antigen-binding arms at a sufficient distance from each other.

[0108] Alternatively, if effector functions are desired, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP) and complement-dependent cytotoxicity (CDC), the multispecific antibody of the invention can further comprise an Fc domain that provides these effector functions. The choice of Fc domain will depend on the type of effector function required.

[0109] In this case, the multispecific antibody of the invention has an immunoglobulin-like structure and comprises:

[0110] - two identical multispecific antigen-binding arms as defined above;

[0111] - dimerized CH2 and CH3 domains of an immunoglobulin;

[0112] - An IgA, IgG or IgD hinge region that links the C-terminus of the CH1 domain of the antigen-binding arm to the N-terminus of the CH2 domain, or when the CH4 domain after the CH3 domain is from IgM or IgE, in which case the C-terminus of the CH1 domain of the antigen-binding arm can be directly linked to the N-terminus of the CH2 domain.

[0113] Preferably, the CH2 and CH3 domains, the hinge region and / or the CH4 domain are derived from the same immunoglobulin as the CH1 domain of the antigen-binding arm or an immunoglobulin having the same isotype and subclass.

[0114] The CH2, CH3 and optionally CH4 domains, as well as the hinge region, from native immunoglobulins can be used. They may also be mutated if desired, for example to modulate the effector functions of the antibody. In some cases, all or part of the CH2 or CH3 domain can be omitted.

[0115] The present invention more specifically provides multispecific, preferably bispecific tetravalent antibodies that contain two binding sites for each of its targets, as well as a functional Fc domain that allows activation of effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis and complement-dependent cytotoxicity (CDC).

[0116] Such preferred antibodies of the present invention are full-length antibodies. They preferably contain heavy and light chains from a human immunoglobulin, preferably IgG, more preferably IgG1.

[0117] The light chain can be a λ or κ light chain, and they are preferably κ light chains.

[0118] In a preferred embodiment, the polypeptide linker for use in the present invention links two pairs of IgG Fab domains in the form of a tetra-Fab multispecific, preferably bispecific antibody, the amino acid sequence of which contains the heavy chain sequences of at least two Fabs linked by a polypeptide linker, followed by a native hinge sequence, followed by an IgG Fc sequence, which is co-expressed with an appropriate IgG light chain sequence.

[0119] Figure 11 Examples of preferred bispecific antibodies of the present invention having an IgG-like structure are shown.

[0120] In a preferred embodiment, the bispecific antibody of the present invention generally comprises

[0121] - A continuous heavy chain consisting of Fc (hinge-CH2-CH3)

[0122] - Subsequently, the continuous Fab heavy chains (CH1-VH) of antibody 1Fab and antibody 2, the latter being linked by a hinge-derived polypeptide linker sequence,

[0123] - And during protein expression, the resulting heavy chains assemble into dimers, while the co-expressed antibody 1 and antibody 2 light chains (VL-CL) bind to their cognate heavy chains to form the final tandem F(ab)'2-Fc molecule.

[0124] Antibody 1 (Ab1) and antibody 2 (Ab2) are different.

[0125] In a preferred embodiment, a bispecific antibody is described, which comprises

[0126] - Two Fab fragments with different mutant CH1 and mutant CL domains, which consist of

[0127] a) A Fab fragment having mutant CH1 and mutant C-κ domains derived from human IgG1 / κ, and the VH and VL domains of Ab1.

[0128] b) A Fab fragment having mutant CH1 and mutant C-κ domains derived from human IgG1 / κ, and the VH and VL domains of Ab2.

[0129] c) A mutant light chain constant domain derived from the human κ constant domain.

[0130] The Fab fragments are arranged in tandem in the following order

[0131] - The C-terminus of the mutant CH1 domain of the Ab1 Fab fragment is linked to the N-terminus of the VH domain of the Ab2 Fab fragment via a polypeptide linker.

[0132] - The hinge region of human IgG1, which links the C-terminus of the mutant CH1 domain of the Ab2 fragment to the N-terminus of the CH2 domain.

[0133] - The dimerization CH2 and CH3 domains of human IgG1.

[0134] Ab1 and Ab2 can be any antibodies for any purpose, especially any antibodies for therapeutic purposes.

[0135] In a particular embodiment, the different Ab1 and Ab2 are independently selected from anti-CD38 antibodies (such as daratumumab) and anti-PD-L1 antibodies (such as atezolizumab).

[0136] In another specific embodiment, the different Ab1 and Ab2 are independently selected from anti-EGFR antibodies and anti-HER2 / neu receptors. In a preferred embodiment, the different Ab1 and Ab2 are independently selected, on the one hand, from cetuximab or its mutant derivatives, and on the other hand, from trastuzumab or its mutant derivatives.

[0137] Such antibodies can be used as drugs, more particularly for the treatment of cancer.

[0138] In a specific example, the bispecific molecule is a bispecific anti-CD38, anti-PD-L1 antibody, which comprises, preferably consists of: a) two heavy chains, each comprising SEQ ID NO: 7, preferably consisting of the same, and b) four light chains, two comprising SEQ ID NO: 15, preferably consisting of the same, and the other two comprising SEQ ID NO: 18, preferably consisting of the same. Such a bispecific antibody is called BiXAb-6567.

[0139] SEQ ID NO: 7 (heavy chain) is:

[0140] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPQAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVDVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0141] This heavy chain sequence contains

[0142] - the VH of daratumumab (SEQ ID NO: 8)

[0143] EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

[0144] - CH1 domain of daratumumab Fab (human IgG1 of G1m(3) allotype, with mutations L124Q and S188V) (SEQ ID NO: 9)

[0145] ASTKGPSVFPQAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV - AP linker (SEQ ID NO: 3)

[0146] EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG

[0147] - VH of atezolizumab (SEQ ID NO: 10)

[0148] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

[0149] - CH1 domain (human IgG1 of G1m(3) allotype of Fab atezolizumab, with mutation T192D (SEQID NO: 11)

[0150] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVDVPSSSLGTQTYICNVNHKPSNTKVDKRV - hinge of human IgG1 (SEQ ID NO: 12)

[0151] EPKSCDKTHTCPPCP

[0152] - CH2 domain of human IgG1 (SEQ ID NO: 13)

[0153] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0154] - CH3 domain of allotype human IgG1 of G1m(3) (SEQ ID NO: 14)

[0155] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0156] The light chain SEQ ID NO: 15 (daratumumab) is EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLVSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0157] This light chain sequence contains

[0158] - VL of daratumumab (SEQ ID NO: 16)

[0159] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0160] - CKappa domain of daratumumab with mutations V133T and S176V (SEQ ID NO: 17) RTVAAPSVFIFPPSDEQLKSGTASVTCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLVSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0161] The light chain SEQ ID NO: 18 (atezolizumab) is DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0162] This light chain sequence contains

[0163] - the VL of atezolizumab (SEQ ID NO: 19)

[0164] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK

[0165] - the CKappa domain of atezolizumab with mutations S114A and N137K (SEQ ID NO: 20)

[0166] RTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0167] The third pair of Fab domains (directed against an antigen target different from the first and second pairs of Fab domains) can also be linked to the same or different polypeptide linkers: i) via the C-terminus of the heavy chain of the third pair of Fab domains to the N-terminus of the heavy chain of an external (i.e., Fc-distal) Fab domain, ii) or via the C-terminus or N-terminus of the heavy chain of the third pair of Fab domains to the C-terminus of the two heavy chains of a Fab domain.

[0168] Any molecule described herein can be modified to include additional non-protein moieties known and readily available in the art, such as by PEGylation or hyperglycosylation. Modifications that can increase serum half-life or stability against proteolytic degradation are included.

[0169] The antibodies of the present invention can be glycosylated or non-glycosylated, or can exhibit multiple glycosylation profiles. In a preferred embodiment, the antibody is non-glycosylated on the variable region of the heavy chain but is glycosylated on the Fc region.

[0170] Humanized forms of reference non-human antibodies can be used. In the humanization method, the complementarity-determining regions (CDRs) from the donor variable region and certain other amino acids are transplanted into a human variable receptor region, which is then linked to a human constant region. See, e.g., Riechmann et al., Nature 332:323-327 (1988); U.S. Patent No. 5,225,539.

[0171] Another example of a construct of the present invention is a bispecific antigen-binding fragment Fab-Fab that does not contain an Fc domain.

[0172] Such Fab-Fab constructs typically contain two different Fab domains. Such antibodies have only one Fab domain each that binds to antigen 1 and antigen 2, respectively. They have the same light chain as the corresponding BiXAb antibody; however, the heavy chains of Fab-Fab are shortened in such a way that their most C-terminal residue is cysteine 220 (according to EU numbering).

[0173] Another example of a construct of the present invention is a dimerized Fab-Fab construct (e.g., via a disulfide bond in the linker or via a native disulfide bond of a hinge at the C-terminus proximal to the Fab domain).

[0174] Design of the linker

[0175] The polypeptide linker, also named "hinge-derived polypeptide linker sequence" or "pseudo-hinge linker", contains all or part of the sequence of the hinge region of one or more immunoglobulins selected from IgA, IgG, and IgD of preferably human origin. The polypeptide linker can contain all or part of the sequence of the hinge region of only one immunoglobulin. In this case, the immunoglobulin can be of the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain is derived, or of a different isotype or subclass. Alternatively, the polypeptide linker can contain all or part of the sequence of the hinge regions of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal part of the polypeptide linker directly after the CH1 domain preferably consists of all or part of the hinge region of an immunoglobulin of the same isotype and subclass as the immunoglobulin from which the CH1 domain is derived.

[0176] Optionally, the polypeptide linker can further contain the sequence of 2 to 15, preferably 5 to 10 N-terminal amino acids of the CH2 domain of an immunoglobulin.

[0177] The polypeptide linker sequence generally consists of less than 80 amino acids, preferably less than 60 amino acids, still preferably less than 40 amino acids.

[0178] In some cases, sequences from natural hinge regions can be used; in other cases, point mutations can be made to these sequences, particularly substituting one or more cysteine residues in the natural IgG1, IgG2, or IgG3 hinge sequences with alanine or serine to avoid unwanted intra-chain or inter-chain disulfide bonds.

[0179] In a specific embodiment, the polypeptide linker sequence comprises the following amino acid sequence or consists of the amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX 10 GG (SEQ ID NO:1), where X1, X2, X3, X4, X5, X6, X7, X8, X9, X10 are the same or different and are any amino acid. In particular, the polypeptide linker sequence can comprise or consist of a sequence selected from the following:

[0180] EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO:2);

[0181] EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO:3);

[0182] EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO:4);

[0183] EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO:5) and EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO:6).

[0184] In a specific embodiment, X1, X2, and X3 are the same or different and are threonine (T) or serine (S).

[0185] In another specific embodiment, X1, X2, and X3 are the same or different and are selected from Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I), still preferably X1, X2, and X3 are the same or different and can be Ala (A) or Gly (G).

[0186] Alternatively, X1, X2, and X3 are the same or different and can 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).

[0187] In certain embodiments, X4 and X5 are the same or different and are any amino acid selected from serine (S), cysteine (C), alanine (A), and glycine (G).

[0188] In preferred embodiments, X4 is serine (S) or cysteine (C).

[0189] In preferred aspects, X5 is alanine (A) or cysteine (C).

[0190] In certain embodiments, X6, X7, X8, X9, X 10 are the same or different and are any amino acid other than threonine (T) or serine (S). Preferably, X6, X7, X8, X9, X10 are the same or different and are selected from Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I).

[0191] Alternatively, X6, X7, X8, X9, X 10 are the same or different and can 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).

[0192] In preferred embodiments, X6, X7, X8, X9, X 10 are the same or different and are selected from Ala (A) and Gly (G).

[0193] In yet another preferred embodiment, X6 and X7 are the same and are preferably selected from Ala (A) and Gly (G).

[0194] In preferred embodiments, the polypeptide linker sequence comprises or consists of the sequence SEQ ID NO: 1, wherein

[0195] X1, X2, and X3 are the same or different and are threonine (T), serine (S);

[0196] X4 is serine (S) or cysteine (C);

[0197] X5 is alanine (A) or cysteine (C);

[0198] X6, X7, X8, X9, X 10 are the same or different and are selected from Ala (A) and Gly (G).

[0199] In another preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence SEQ ID NO: 1, wherein

[0200] X1, X2 and X3 are the same or different and are Ala (A) or Gly (G);

[0201] X4 is serine (S) or cysteine (C);

[0202] X5 is alanine (A) or cysteine (C);

[0203] X6, X7, X8, X9, X 10 are the same or different and are selected from Ala (A) and Gly (G).

[0204] In the case of a multispecific antigen-binding fragment comprising more than two different Fab fragments, the polypeptide linkers separating the Fab fragments can be the same or different.

[0205] Generation of multispecific antibodies:

[0206] Those skilled in the art can refer to International Patent Application WO2013 / 005194, the general techniques for expressing multispecific antibodies of which are incorporated herein by reference.

[0207] In addition, polynucleotides comprising sequences of protein chains encoding the molecules or antibodies of the present invention are described herein. The polynucleotides may further comprise additional sequences: specifically, they may advantageously comprise sequences encoding a leader sequence or signal peptide that allows secretion of the protein chain. Host cells transformed with the polynucleotides are also disclosed.

[0208] Generally, optionally after codon optimization for mammalian expression, the amino acid sequences of different monoclonal antibodies are used to design the DNA sequences. For the heavy chain, synthetic DNA is made which encodes a signal peptide, the variable region of Fab1 and the constant CH1 domain, followed by a hinge linker and the variable region and constant CH1 domain of Fab2 and flanking sequences for restriction enzyme digestion. For the light chain, synthetic DNA is made which encodes a signal peptide and the variable and constant κ regions.

[0209] Nucleic acids encoding the heavy and light chains of the antibodies of the present invention are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of the immunoglobulin polypeptides. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. The expression vector usually replicates in the host organism as an episome or as an integrated part of the host chromosomal DNA. Usually, the expression vector will contain a selectable marker (such as tetracycline or neomycin) to allow the detection of those cells transformed with the desired DNA sequence.

[0210] In one example, both the heavy and light chain coding sequences (e.g., sequences encoding VH and VL, VH-CH1 and VL-CL, or full-length heavy chain and full-length light chain) are contained in one expression vector. In another example, each heavy and light chain of the antibody is cloned into a separate vector. In the latter case, the expression vectors encoding the heavy and light chains can be co-transfected into a host cell to express both chains, which can be assembled in vivo or in vitro to form a complete antibody. Alternatively, the expression vector encoding the heavy chain and / or those encoding the light chain can be introduced into different host cells to express each of the heavy and light chains, which can then be purified and assembled to form a complete antibody in vitro.

[0211] In a particular embodiment, a host cell is co-transfected with three independent expression vectors (such as plasmids), resulting in the co-production of all three chains (designated as heavy chain HC and two light chains LC1 and LC2, respectively), and the secretion of a multispecific, e.g., bispecific, antibody.

[0212] More particularly, these three vectors can advantageously be used in a molar ratio of 2:1:1 (HC:LC1:LC2).

[0213] A host cell transfected with an expression vector containing a polynucleotide encoding a heavy chain as defined herein can be further transformed with at least two polynucleotides encoding two different light chains: a first light chain that specifically pairs with the first VH / CH1 region of the heavy chain; a second light chain that specifically pairs with the second VH / CH1 region of the heavy chain.

[0214] In other embodiments, the host cell can be additionally transformed with a polynucleotide encoding a third light chain that is different from the first and second light chains and specifically pairs with the third VH / CH1 region of the heavy chain.

[0215] Recombinant vectors for expressing the antibodies described herein typically contain nucleic acids encoding antibody amino acid sequences operably linked to a constitutive or inducible promoter. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the nucleic acids encoding the antibody. The vector optionally contains a universal expression cassette that includes at least one independent terminator sequence, sequences that permit replication of the cassette in eukaryotes and prokaryotes, i.e., a shuttle vector, and selectable markers for prokaryotic and eukaryotic systems.

[0216] Multispecific, e.g., bispecific, antibodies as described herein can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, plants, insects (e.g., using baculovirus vectors), and mammalian cells. The recombinant antibodies of the invention need not be 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 (293 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 hepatocytes (Hep G2 cells).

[0217] Mammalian tissue cell cultures are preferred for expressing and producing polypeptides because many suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines (e.g., NS0) or transformed B cells or hybridomas.

[0218] In a most preferred embodiment, the multispecific, e.g., bispecific, antibodies of the invention are produced by using a CHO cell line, most advantageously the CHO-S or CHO-DG-44 cell line or derivatives thereof.

[0219] Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, and enhancers, as well as essential processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, and the like.

[0220] Vectors containing the target polynucleotide sequences (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary according to the type of cell host. For example, calcium phosphate treatment or electroporation can be used for other cell hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). When the heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of the complete immunoglobulin.

[0221] Transform or transfect host cells with the vector (e.g., by chemical transfection or electroporation methods), and culture them in a conventional nutrient medium (or a modified medium as appropriate) to induce the promoter, select transformants or amplify the gene encoding the desired sequence.

[0222] Expression of the antibody can be transient or stable.

[0223] Preferably, multispecific, e.g., bispecific antibodies, are produced by a method of stable expression, in which a cell line stably transfected with DNA encoding all polypeptide chains of the multispecific, e.g., bispecific antibody, e.g., BiXAb-6567, can continuously express, which enables the preparation of therapeutic agents. For example, stable expression in a CHO cell line is particularly advantageous.

[0224] Once expressed, the complete antibodies, their dimers, individual light and heavy chains or other immunoglobulin forms of the present invention can be further isolated or purified to obtain a substantially homogeneous preparation for further assays and applications. Standard protein purification methods known in the art can be used. For example, suitable purification procedures can include fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation and gel filtration (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982)). For pharmaceutical use, a substantially pure composition with at least about 90 - 95% homogeneity is preferred, and most preferably 98 - 99% or higher homogeneity.

[0225] In vitro production allows for scale-up to produce large amounts of the desired multispecific, e.g., bispecific antibodies of the present invention. Such methods can employ homogeneous suspension culture in, for example, an airlift reactor or a continuously stirred reactor, or fixed or entrapped cell culture on agarose microbeads or ceramic cartridges, e.g., in hollow fibers, microcapsules.

[0226] Application

[0227] The protein constructs or bispecific antibodies of the present invention can be used in all applications of bispecific antibodies. In particular, they can be used to obtain such medicaments which are used in patients (conjugated with radioactive, fluorescent, chemiluminescent or any other label) or in vitro (e.g., for cell and tissue staining using immunohistochemistry, immunoblotting and immunofluorescence techniques) for a wide range of therapeutic and diagnostic medical applications. These medicaments or diagnostic products are also part of the object of the present invention.

[0228] Other aspects of the present invention are pharmaceutical compositions which comprise a protein construct or an antibody according to the present invention. Another aspect of the present invention is the use of a protein construct or an antibody according to the present invention in the preparation of a pharmaceutical composition. Other aspects of the present invention are methods for manufacturing a pharmaceutical composition comprising a protein construct or an antibody according to the present invention.

[0229] In another aspect, the present invention provides a composition comprising a protein construct or an antibody as defined herein formulated together with a pharmaceutical carrier, such as a pharmaceutical composition.

[0230] The compositions of the present invention can be administered by a variety of methods known in the art.

[0231] The following examples are provided by way of illustration and are not intended to limit the present invention. Examples

[0232] Example 1: Preparation of the bispecific antibody BiXAb-6567 of the present invention

[0233] Gene synthesis

[0234] After codon optimization for mammalian expression, DNA sequences were designed using the GeneScript program with the amino acid sequences of anti-CD38 (daratumumab) and anti-PDL1 (atezolizumab). These antibodies are referred to as the "parent" anti-CD38 and "parent" anti-PD-L1 mAbs.

[0235] The DNA construct of the heavy chain was designed as follows: a signal peptide, followed by the sequence SEQ ID NO: 7 [consisting of a variable region, followed by the constant CH1 domain of Fab1 (anti-CD38), where mutations Leu to Gln and Ser to Val were introduced at Kabat positions 124 and 188 respectively, followed by a linker, then a variable region, followed by the constant CH1 domain of Fab2 (anti-PD-L1), where a mutation Thr to Asp was introduced at Kabat position 192]; flanking sequences for restriction enzyme digestion were introduced at both ends of the heavy chain DNA construct. The DNA construct of the light chain was designed as follows: a signal peptide (SEQ ID NO: 21), followed by a variable region, followed by the constant κ region. For the anti-CD38 light chain (SEQ ID NO: 15), mutations were introduced at Kabat positions 133 (Val to Thr) and 176 (Ser to Val) in the constant κ domain. For the anti-PDL1 light chain (SEQ ID NO: 18), mutations at Kabat positions 114 (Ser to Ala) and 137 (Asn to Lys) were introduced into the constant κ domain. All DNA constructs were synthesized by Gene Art.

[0236] PCR reactions were performed using PfuTurbo Hot Start to amplify the inserts, and then the inserts were digested with NotI and ApaI, and NotI and HindIII for the heavy and light chains respectively. The double-digested heavy chain fragment was ligated to the pcDNA3.1 expression vector (Invitrogen) treated with NotI and ApaI, into which the human IgG1 hinge and CH2-CH3 domains had already been inserted. The double-digested light chain fragment was ligated to the pcDNA3.1 expression vector (Invitrogen) treated with NotI and HindIII. The plasmid DNA was verified by double-stranded DNA sequencing.

[0237] Expression and purification

[0238] In serum-free medium in an adapted suspension (CHO SFM-II medium, Life Technologies TM) In [the experiment], bispecific antibody BiXAb-6567 was generated using transient gene expression by co-transfecting 3 genes encoded on separate vectors at a molecular ratio of 2:1:1 = HC:LC1:LC2 (1 continuous heavy chain (HC) and 2 light chains (LC)). Generally, for a 50 mL scale expression, a total of 50 μg plasmid DNA (25 μg heavy chain, 12.5 μg anti-CD38 light chain, and 12.5 μg anti-PD-L1 light chain) was mixed in a 1.5 mL Eppendorf tube, and then 1 mL of CHO SFM medium containing 25 μL of 3 mg / mL PEI transfection reagent pH 7.0 (Polyplus) was added, and the reaction was incubated at room temperature for 20 minutes. Subsequently, the DNA-PEI mixture was added to 49 mL of Invitrogen FreeStyle from Life Technologies in a 125 mL shake flask TM CHO-S cells (1 - 2 x 10 6 / mL). The cells were shaken for 6 days. The supernatant was collected by centrifugation at 3,000 rpm for 15 minutes. The expression titer of BiXAb-6567 in the supernatant was determined using a Protein A biosensor from ForteBio Systems). Then BiXAb-6567 was purified on a Protein A affinity resin (MabSelect SuRe, GE Healthcare Life Sciences). The antibody was eluted from Protein A using 0.1 M glycine pH 3.5, and the eluate was neutralized with 1 M TRIS. The purified antibody in Dulbecco's PBS (Lonza) was sterile filtered (0.2 μM sterile filter, Techno Plastic Products AG), and the final concentration was determined by reading the optical density (OD) at 280 nm (Eppendorf ).

[0239] BiXAab-6567 generally shows good expression titers (>180 mg / L) in transient CHO expression. This expression level is comparable to that seen for conventional monoclonal antibodies.

[0240] SDS polyacrylamide gel electrophoresis

[0241] To evaluate the quality of the purified BiXAb-6567, we performed SDS-PAGE (Experion TMAutomated electrophoresis system, BioRad). In the presence of sodium dodecyl sulfate (SDS) in the running buffer, the rate at which antibodies migrate in the gel depends mainly on their size, allowing the molecular weight to be determined. This assay is performed under non-reducing and reducing conditions; the latter allows the disruption of disulfide bonds and thus the visualization of individual polypeptide chains (light and heavy chains).

[0242] SDS-PAGE data are presented in Figure 1 . Under non-reducing conditions, the quaternary structure of the antibody is maintained, and the observed molecular weight should represent the sum of the molecular weights of the different heavy and light chains. The bispecific antibody of the present invention (BiXAb-6567) consists of six chains: two heavy chains and four light chains. The theoretical molecular weight of BiXab-6567 is 244.40 kDa, without considering post-translational modifications (PTMs), such as N-glycosylation in the Fc at asparagine 297. The gel is calibrated using a mixture of standards of known molecular weight. The non-reducing data show a main band close to the 250 kDa molecular weight standard, which is consistent with the calculated molecular weight and the expected glycosylation of the two asparagines at position 297 of the Fc domain. Under reducing conditions, dithiothreitol (DTT) further denatures BiXAb-6567 by reducing disulfide bonds and disrupting the quaternary structure, so that the six polypeptide chains should migrate separately in the gel according to their molecular weights. The two identical heavy chains of BiXAb-6567 co-migrate as one band, while the two pairs of light chains co-migrate as a second band due to their almost identical molecular weights. Thus, based on the mobility relative to the molecular weight standards, the data show two main bands at approximately 75 kDa and 25 kDa. Each heavy chain has a single N-glycosylation site at asparagine 297, which accounts for the width of the higher molecular weight band and the observed molecular weight being slightly higher than the calculated molecular weight (75.44 kDa); this broadening is typical for glycosylated proteins. The calculated molecular weights of the anti-CD38 (23.40 kDa) and anti-PD-L1 (23.36 kDa) light chains are very similar, resulting in their co-migration.

[0243] In summary, when considering the presence of N-glycosylation sites in the heavy chains, the SDS-PAGE of BiXAb-6567 shows the expected profiles under both non-reducing and reducing conditions and is consistent with the calculated theoretical molecular weights.

[0244] Size exclusion chromatography analysis

[0245] Protein aggregation is frequently observed in engineered protein molecules. We performed analytical size exclusion chromatography (SEC) to determine the high molecular weight species content of the one-step affinity purified BiXAb-6567 preparation (see Expression and purification of variants). We used an SEC-s3000 (300x 7.8 mm) column (BioSep) and an Akta purifier 10 system (GE Healthcare); the determination was performed at a flow rate of 1 mL / min using PBS buffer pH 7.4.

[0246] Figure 2 The SEC chromatogram presented in Figure 4 demonstrates that the main peak corresponds to the expected size of the monomeric BiXAb-6567; this peak accounts for 98.2% of the total sample. In addition, a small peak was observed, corresponding to a higher molecular weight species (presumably a dimer); this peak accounts for 1.8% of the total sample. Therefore, we conclude that the percentage content of the higher molecular weight species is small and is similar to that of conventional monoclonal antibodies produced in the CHO expression system. The narrow and symmetric shape of the monomer peak suggests that BiXAb-6567 has been correctly assembled and is represented by a single species.

[0247] Example 2: Characterization of BiXAb-6567 by differential scanning calorimetry (DSC)

[0248] Differential scanning calorimetry (DSC) was used to compare the thermal stabilities of BiXAb-6567, the parental anti-CD38 mAb, and the parental anti-PD-L1 mAb. A MicrocalTM VP-Capillary DSC system (Malvern Instruments) was used to perform the differential scanning calorimetry experiments.

[0249] All samples were centrifuged (20,000 x g, 5 minutes, 4 °C), and their protein content was quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) with an IgG analysis program before DSC analysis. For the determination, all samples were diluted to a final concentration of 1 mg / mL in PBS

[0250] The pre-equilibration time was 3 minutes, and the resulting thermograms were obtained at a scan rate of 60 °C / h between 20 and 110 °C, with a filter time of 25 seconds and medium feedback. Five buffer / buffer scans were measured to stabilize the instrument before sample analysis, and buffer / buffer scans were performed between each protein / buffer scan. The data conformed to a non-two-state unfolding model and were adjusted by subtracting the baseline before and after the transition.

[0251] Figure 3The DSC curves presented herein (covering the range of 50 to 100 °C) demonstrate the manner in which each Fv region can result in a different Fab unfolding profile; this experiment also demonstrates that the Fv region determines the apparent stability of the Fab. The DSC spectrum of the anti-CD38 mAb shows two transitions: a large peak with a Cp max of 170 Kcal / mole / °C and a Tm1 of 70.9 °C, corresponding to the unfolding of both the CH2 and Fab domains, and a small peak with a Cp max of 20 Kcal / mole / °C and a Tm2 of 81.5 °C, corresponding to the unfolding of the CH3 domain. The DSC spectrum of the anti-PD-L1 mAb shows two transitions: a small peak with a Cp max of 20 Kcal / mole / °C and a Tm1 of 69.9 °C, corresponding to the unfolding of the CH2 domain, and a large peak with a Cp max of 160 Kcal / mole / °C and a Tm2 of 83.4 °C, corresponding to the unfolding of the CH3 and Fab domains.

[0252] The DSC spectrum of BiXAb-6567 also shows two transitions with two large peaks. The first peak has a Cp max of 130 Kcal / mole / °C and a Tm1 of 71.5 °C and corresponds to the unfolding of the CH2 and Fab domains of the anti-CD38 mAb. The second peak has a Cp max of 170 Kcal / mole / °C and a Tm2 of 81.5 °C and corresponds to the unfolding of the CH3 and Fab domains of the anti-PD-L1 mAb. Thus, the DSC spectrum of BiXAb-6567 is similar to the superposition of the two DSC spectra of the two parental mAbs and illustrates the excellent assembly and stability of BiXAb-6567. The Tonset of BiXAb-6567 (63.3 °C) is similar to that of the parental mAbs (anti-CD38 Tonset = 63.5 °C, while anti-PD-L1 Tonset = 63.2 °C), indicating that BiXAb-6567 has stability properties similar to those of the parental antibodies. The calculated ΔH of BiXAb-6567 is 1560 kcal / mol, reflecting the larger size of the bispecific molecule relative to the two parental antibodies (anti-CD38 ΔH = 963 kcal / mol, while anti-PD-L1 ΔH = 820 kcal / mol).

[0253] Definition:

[0254] Tm or denaturation / melting temperature is the point at which the concentrations of the unfolded and folded species are equal and is the midpoint of the unfolding transition. As a parameter, it describes the sensitivity of the protein to thermal denaturation and is thus related to the stability of the protein. The higher the Tm, the more stable the protein.

[0255] Tonset is the temperature at which the unfolding transition begins. The value of this parameter is typically 5 to 10 °C lower than Tm. It is also a parameter that describes protein stability, but is related to resistance to thermal denaturation.

[0256] ΔH is the calorimetric enthalpy of unfolding and reflects the disruption of intramolecular interactions in the protein (i.e., disruption of interactions within and between domains). The thermal unfolding process is endothermic and thus results in a positive enthalpy value. The calorimetric enthalpy (ΔH) is the area under the peak of the thermal unfolding transition.

[0257] Example 3: Cell-free Binding Properties of BiXAb-6567

[0258] Direct CD38 antigen-binding plate ELISA assay

[0259] 100 μl of each parental mAb, anti-CD38 or anti-PDL1, each at a concentration of 3 μg / mL prepared by dilution with PBS pH 7.4, was used to coat Maxisorp plates overnight at 4 °C. Additionally, BiXAb-6567 at a concentration of 5 μg / mL prepared by dilution with PBS pH 7.4 was used to coat Maxisorp plates overnight at 4 °C. The plates were washed 5 times with 1x PBS containing 0.05% Tween-20 (PBST), and then blocked with 1% BSA in 1x PBS at 200 μL / well for 200 hours at room temperature. Subsequently, the plates were washed 5 times with 1x PBST. A 7-point 3-fold dilution series of recombinant CD38 His / Flag-tagged (CreativeBiomart) starting at 1 μg / mL in 1x PBS was prepared; 100 μL of each dilution step was added to each assay well. The plates were incubated for 1 hour at room temperature and washed 5 times with 1x PBST. 100 μL / well of anti-Flag-tag antibody-conjugated HRP (Abcam) diluted 10,000-fold in 1x PBS was added, and the plates were incubated for 1 hour at room temperature. After washing 5 times with 1x PBST, 100 μL / well of TMB substrate in 1x PBS was added for colorimetric reading, and the plates were incubated for 15 minutes at room temperature to develop color. Assay data was collected at 650 nm using a Victor2 microplate reader (Perkin Elmer).

[0260] BiXAb-6567 exhibited a dose-dependent binding curve very similar to that of the parental anti-CD38 antibody ( Figure 4A)。The EC50 values for the binding of CD38 to the two antibodies are as follows: EC50[BiXAb-6567] = 171 ng / mL, while EC50[anti-CD38] = 199 ng / mL. This result indicates that BiXAb-6567 has a correctly assembled anti-CD38 Fab domain, as it exhibits binding similar to that of the parental anti-CD38 mAb. As expected, the parental anti-PDL1 mAb used as a negative control did not show any binding.

[0261] Direct PDL1 antigen-binding plate ELISA analysis.

[0262] 100 μL of biotinylated human PD-L1 protein (AcroBiosystems) at a concentration of 1 μg / mL prepared by dilution with 1x PBS pH 7.4 was used to coat Maxisorp plates overnight at 4°C. The plates were washed 5 times with PBST and then blocked with 1% BSA in 1x PBS at 200 μL / well for 2 hours at room temperature. Subsequently, the plates were washed 5 times with 1x PBST. A 7-point 3-fold dilution series of anti-CD38 mAb (starting at 0.3 mg / mL) or anti-PD-L1 mAb (starting at 0.3 mg / mL) or BiXAb-6567 (starting at 0.5 mg / mL) in 1x PBS solution was prepared; 100 μL of each dilution step was added to each assay well. The plates were incubated for 1 hour at room temperature and washed 5 times with 1x PBST. 100 μL / well of anti-human antibody (IgG H&L) conjugated HRP (Abliance) diluted 5,000-fold in 1x PBS was added, and the plates were incubated for 1 hour at room temperature. After washing 5 times with 1x PBST, 100xL / well of TMB substrate in 1x PBS was added for colorimetric reading, and the plates were incubated for 15 minutes at room temperature to develop color. Assay data were collected at 650 nm using a Victor2 microplate reader (Perkin Elmer).

[0263] BiXAb-6567 exhibited a dose-dependent binding curve very similar to that of the parental anti-PD-L1 antibody ( Figure 4B )。The EC50 values for the binding of PD-L1 to the two antibodies are as follows: EC50[BiXAb-6567] = 93 ng / mL, while EC50[anti-PD-L1] = 72 ng / mL. This result indicates that BiXAb-6567 has a correctly assembled anti-PD-L1 Fab domain, as it shows binding similar to that of the parental anti-PD-L1 mAb. As expected, the parental anti-CD38 mAb used as a negative control did not show any binding.

[0264] Dual antigen-binding ELISA assay

[0265] 100 μL of 2 μg / mL recombinant human Fc-tagged CD38 (CreativeBioMart), prepared by dilution with 1x PBS pH 7.4, was used to coat Maxisorp plates overnight at 4 °C. The plates were washed 5 times with 1x PBST and then blocked with 1% BSA in 1x PBS at 200 μL / well for 2 hours at room temperature. The plates were washed 5 times with 1x PBST. A 7-point 3-fold dilution series of BiXAb-6567 in 1x PBS (starting at 1 μg / mL) was prepared and 100 μL of each dilution step was added to each assay well. The plates were incubated for 1 hour at room temperature, followed by 5 washes with 1x PBST. 100 μL / well of 1 μg / mL biotinylated human PD-L1 (AcroBiosystems) in 1x PBS was added and the plates were incubated for 1 hour at room temperature. After washing 5 times with 1x PBST, 100 μL / well of 0.1 μg / mL streptavidin-conjugated HRP (Biotechne), prepared by dilution with 1x PBS, was added. The plates were incubated for 1 hour at room temperature. After washing 5 times with 1x PBST, 100 μL / well of TMB substrate in 1x PBS was added for colorimetric reading and the plates were incubated for 15 minutes at room temperature for color development. Assay data were collected at 650 nm using a Victor2 microplate reader (Perkin Elmer).

[0266] BiXAb-6567 exhibited a dose-dependent binding curve in the dual ELISA format, indicating that it has correctly assembled anti-CD38 and anti-PD-L1 Fab domains( Figure 4C ). This indicates that BiXAb-6567 is a bispecific antibody that is capable of binding both CD38 and PD-L1 simultaneously with an EC50 = 144 ng / mL. As expected, neither of the parental mAbs, anti-CD38 or anti-PDL1, showed any binding in this dual ELISA format.

[0267] Example 4: Determination of relative binding activity by fluorescence-activated cell sorting (FACS)

[0268] CHO-CD38 cells (CHO cells stably transfected with full-length human CD38) were cultured in DMEM-Glutamax-I medium supplemented with 100 μg / ml penicillin, 100 μg / ml streptomycin, 10% fetal bovine serum, and 500 μg / ml geneticin. SKOV-3 cells and RPMI-8226 cells were cultured in RPMI 1640-Glutamax-I medium supplemented with 100 μg / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum.

[0269] For each sample, 3x105 cells (CHO-CD38 or SKOV-3 or RPMI-8226) were used. The cells were washed once with PBA solution (PBS supplemented with 1% BSA and 0.05% sodium azide). To determine the FACS profile, the cells were stained with each antibody at a concentration of 50 μg / ml in a volume of 30 μl. To titrate BiXAb-6567 and the parental anti-CD38 antibody, and subsequently determine the binding parameters, CHO-CD38 cells were stained with each antibody at the indicated concentrations in a volume of 30 μl. The cells were incubated on ice for 30 minutes and then washed twice with 1 ml of PBA solution. The cells were incubated with a fluorescently labeled anti-human κ or anti-human IgG Fcγ-specific secondary antibody in the dark on ice for 30 minutes and then washed twice with 1 ml of PBA solution; finally, the cells were resuspended in PBA solution with a final volume of 500 μl. Samples were analyzed using an Epics-XL or Navios flow cytometer (Beckman Coulter). 10,000 events were obtained in each experiment.

[0270] The binding profiles of BiXAb-6567 with the parental anti-CD38 and anti-PD-L1 parental antibodies are presented in Figure 5A -C. We chose to test the multiple myeloma cell line RPMI-8226, which expresses high levels of CD38 and negligible levels of PD-L1 ( Figure 5A ); the CHO-CD38 cell line, which expresses very high levels of CD38 due to stable transfection with full-length CD38 ( Figure 5B ); and the ovarian cancer cell line SKOV-3, which is known to express PD-L1 ( Figure 5C ). These profiles showed a single peak for BiXAb-6567, which was very similar to the profiles of the two parental antibodies on the 3 cell lines. This indicates that BiXAb-6567 is correctly folded and has binding properties similar to those of the parental antibodies. As expected, CHO-CD38 expresses only CD38 and not PD-L1, while SKOV-3 expresses only PD-L1 and not CD38.

[0271] To quantitatively confirm that the binding properties of BiXAb-6567 are similar to those of the parental anti-CD38 antibody, titrations of BiXAb-6567 and the anti-CD38 parental antibody were performed using CHO-CD38 cells, as Figure 6 presented. The EC50 of BiXAb-6567 was determined to be 17.1 nM, while the EC50 of the parental anti-CD38 was 8.5 nM, confirming the similar binding properties of the anti-CD38 Fab domains in BiXAb-6567 and the parental anti-CD38 antibody. As expected, the negative controls (anti-PD-L1 and anti-CD20 antibodies) in this experiment did not demonstrate binding to CHO-CD38 cells.

[0272] Example 5: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Using Unfractionated, Non-Preactivated Mononuclear Cells (MNCs)

[0273] Culture CHO-CD38, SKOV-3, and RPMI-8226 cells as described in Example 4 above.

[0274] To prepare MNCs, the following procedure was employed. Freshly drawn peripheral blood was anticoagulated with citrate. Subsequently, 5 ml of Ficoll-Paque PLUS solution was layered over 6 ml of anticoagulated whole blood. The sample was centrifuged at 2500 rpm for 20 minutes at room temperature, followed by no centrifuge breakage. MNCs were collected from the plasma / Ficoll interface. The MNC cell suspension was diluted 1:10 in PBS and centrifuged at 1800 rpm for 5 minutes at room temperature. The supernatant was removed, and red blood cells were lysed by adding 45 ml of ice-cold distilled water to the cell suspension for 30 seconds, followed by the addition of 5 ml of 10x PBS. The cells were centrifuged at 1800 rpm for 5 minutes at room temperature and washed 3 times with 1x PBS to remove platelets. Finally, the cells were resuspended in 5 ml of cell culture medium. The cell number was adjusted in the ADCC assay to achieve a 40:1 = effector cell:tumor cell ratio.

[0275] For ADCC 51 chromium release assay, 1 x 10 6 target cells (RPMI 8226, SKOV-3, or CHO-CD38) were incubated with 100 μCi of 51 chromium in 200 μl of PBS at 37 °C and 5% CO2 for 2 hours. After the 2-hour incubation, the cells were washed 3 times with 7 ml of medium and finally resuspended at a concentration of 0.1 x 106 cells / ml. The target cells (5,000 cells / well) and MNCs were incubated in the presence of antibody in a 96-well microtiter plate (200 μl assay volume) at 37 °C and 5% CO2 for 3 hours. To determine the maximum target cell lysis (= maximum cpm), Triton X-100 was added. To determine the basal 51 chromium release (= basal cpm), the target cells were not further processed. After 4 hours of incubation, the microtiter plate was centrifuged at 2000 rpm for 5 minutes, and 25 μl of the supernatant was mixed with 125 μl of Optiphase Supermix (Perkin Elmer) and incubated in an oscillating incubator for 1 minute. The samples were assayed in a MicroBeta TriLux (Perkin Elmer) β-counter instrument. Target cell lysis was calculated using the following formula:

[0276] Lysis percentage = (experimental cpm - basal cpm) / (maximum cpm - basal cpm) x 100.

[0277] All measurements were performed in triplicate.

[0278] ADCC assays of CD38+ cells (RPMI-8226 and CHO-CD38) were performed using non-pre-activated MNCs as effector cells ( Figure 7 and 8 ). The assays showed that BiXAb-6567 and the anti-CD38 antibody, which had EC50 values of 0.8 nM and 0.3 nM, respectively, against RPMI-8226 cells, had potent cytotoxicity; on CHO-CD38 cells, the cytotoxicities of BiXAb-6567 and the anti-CD38 antibody had EC50 values of 0.2 nM and 0.07 nM, respectively. Anti-PD-L1 showed minimal activity in both cell lines; as expected, two negative control mAbs (anti-CD20 and anti-HER2) did not promote any lysis. These results demonstrated the potent ADCC activity of BMX-6567 against CD38+ cells, which was similar to the activity of the parental anti-CD38 antibody.

[0279] Example 6: ADCC using enriched pre-activated NK cells

[0280] SKOV3 cells, RPMI 8226 and CHO-CD38 cells were cultured as described in Example 4. MNCs were prepared as described in Example 5. NK cells were isolated from MNCs by negative selection using the "NK Cell Isolation Kit, human" (Miltenyi) according to the manufacturer's instructions. NK cells were cultured overnight at an inoculation density of 2 x 10 6 cells / ml in RPMI medium supplemented with 10% fetal bovine serum. IL-12 or IL-15 was added to a final concentration of 10 ng / ml. The ADCC assay was performed as outlined in Example 5, except that the effector cell:tumor cell ratio was maintained at 10:1 and the reaction duration was reduced to 3 hours.

[0281] The ADCC properties of the anti-PD-L1 portion of BiXAb-6567 were assayed using IL-12 or IL-15 pre-activated enriched NK cells on the PD-L1+ cell line SKOV-3. The results are presented in Figure 9 and 10 . This experiment compared the ADCC properties of BiXAb-6567 with those of the parental anti-PD-L1 antibody; since SKOV-3 cells are PD-L1+ / HER2+ / CD20- / CD38-, the anti-HER2 antibody was used as a positive control, and the anti-CD20 antibody and the parental anti-CD38 antibody were used as negative controls.Figure 9 and 10 demonstrated potent ADCC activity of BiXAb-6567 and the parental anti-PD-L1 antibody, independent of the use of IL-12 or IL-15 in cultured NK cells. When using IL-12, the EC50 values of BiXAb-6567 and the parental anti-PD-L1 antibody were 0.007 nM and 0.03 nM, respectively. When using IL-15, the situation was even more similar; however, curve fitting did not converge, thus precluding the calculation of EC50 values. These results demonstrated potent ADCC activity of BMX-6567 against PD-L1+ cells, which was similar to the ADCC activity of the parental anti-PD-L1 antibody. Sequence Listing <110> BIOMUNEX Pharmaceuticals <120> Stable Bispecific Antibody <130> B2466 <160> 21 <170> PatentIn version3.5 <210> 1 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Linker Sequence 1 <220> <221> misc_feature <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (8)..(8) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (10)..(11) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (14)..(14) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (24)..(25) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (28)..(28) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (30)..(30) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (32)..(32) <223> Xaa can be any naturally occurring amino acid <400> 1 Glu Pro Lys Xaa Cys Asp Lys Xaa His Xaa Xaa Pro Pro Xaa Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Xaa Xaa Pro Pro Xaa Pro Xaa Pro Xaa 20 25 30 Gly Gly <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Linker Sequence 2 <400> 2 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Gly Gly Pro Pro Gly Pro Gly Pro Gly 20 25 30 Gly Gly <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Linker sequence 3 <400> 3 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ala Ala Pro Pro Ala Pro Ala Pro Ala 20 25 30 Gly Gly <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Linker sequence 4 <400> 4 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ala Ala Pro Pro Gly Pro Ala Pro Gly 20 25 30 Gly Gly <210> 5 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Linker sequence 5 <400> 5 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser 20 25 30 Gly Gly <210> 6 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Linker Sequence 6 <400> 6 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser 20 25 30 Gly Gly <210> 7 <211> 702 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Gln Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Val Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ala Ala Pro Pro Ala Pro Ala Pro Ala Gly Gly Glu Val 245 250 255 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 260 265 270 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile 275 280 285 His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp 290 295 300 Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 305 310 315 320 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 325 330 335 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 340 345 350 Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 355 360 365 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 370 375 380 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 385 390 395 400 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 405 410 415 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 420 425 430 Gly Leu Tyr Ser Leu Ser Ser Val Val Asp Val Pro Ser Ser Ser Leu 435 440 445 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 450 455 460 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 465 470 475 480 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 485 490 495 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 500 505 510 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 515 520 525 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 530 535 540 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 545 550 555 560 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 565 570 575 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 580 585 590 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 595 600 605 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 610 615 620 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 625 630 635 640 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 645 650 655 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 660 665 670 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 675 680 685 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 690 695 700 <210> 8 <211> 122 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 98 <212> PRT <213> Artificial sequence <220> <223> CH1 of daratumumab <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Gln Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Val Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 10 <211> 118 <212> PRT <213> Artificial sequence <220> <223> VH of atezolizumab <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 11 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> CH1 of atezolizumab <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Asp Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 12 <211> 15 <212> PRT <213> Homo sapiens <400> 12 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 13 <211> 110 <212> PRT <213> Homo sapiens <400> 13 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 14 <211> 107 <212> PRT <213> Homo sapiens <400> 14 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 15 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LC daratumumab <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Thr Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Val 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 107 <212> PRT <213> Homo sapiens <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Ckappa of daratumumab <400> 17 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Thr Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Val Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 18 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LC atezolizumab <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ala Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Lys Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL atezolizumab <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Ckappa atezolizumab <400> 20 Arg Thr Val Ala Ala Pro Ala Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Lys Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 21 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Signal Peptide <400> 21 Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly 1 5 10 15 Val Gln Cys

Claims

1. A protein construct comprising multi-specific antigen-binding fragments, said multi-specific antigen-binding fragments comprising at least two Fab fragments having different CH1 and CL domains, wherein each Fab fragment recognizes a different epitope of interest, and said Fab fragments are arranged in series in any order, the C-terminal end of the CH1 domain of the first Fab fragment is linked to the N-terminal end of the VH domain of the subsequent Fab fragment by a polypeptide linker, wherein the two Fab fragments are mutant Fab fragments defined as follows: a) A Fab fragment consisting of: - the VH and VL domains of an antibody of interest; - a CH1 domain derived from said CH1 domain by substituting the threonine residue at position 192 of the CH1 domain of human IgG1 immunoglobulin with an aspartic acid residue, and - a kappa-type CL domain derived from said CL domain by substituting the asparagine residue at position 137 of the CL domain of human immunoglobulin with a lysine residue and substituting the serine residue at position 114 of said CL domain with an alanine residue; and b) A Fab fragment consisting of: - the VH and VL domains of an antibody of interest; - a CH1 domain derived from said CH1 domain by substituting the leucine residue at position 124 of the CH1 domain of human IgG1 immunoglobulin with a glutamine residue and substituting the serine residue at position 188 of said CH1 domain with a valine residue; and - a kappa-type CL domain derived from said CL domain by substituting the valine residue at position 133 of the CL domain of human immunoglobulin with a threonine residue and substituting the serine residue at position 176 of said CL domain with a valine residue; wherein the sequence position numbers used herein for the CH1 and CL domains are Kabat numbers.

2. The protein construct of claim 1, which is a multi-specific antibody having two identical antigen-binding arms, each arm consisting of a multi-specific antigen-binding fragment as defined in claim 1.

3. The protein construct of claim 2, which has an immunoglobulin-like structure, comprising: - two identical antigen-binding arms, each arm consisting of a multi-specific antigen-binding fragment as defined in claim 1; - the dimerized CH2 and CH3 domains of an immunoglobulin; - a hinge domain of IgA, IgG or IgD that links the C-terminal end of the CH1 domain of said antigen-binding arm to the N-terminal end of said CH2 domain.

4. The protein construct of claim 3, which is a bispecific antibody and comprises at least two heavy chains and four light chains, wherein each heavy chain comprises a. an Fc region of an immunoglobulin comprising a hinge-CH2-CH3 domain, b. said Fc region is linked to the Fab heavy chain CH1-VH of antibody 1 (Ab1) through said hinge domain, c. It is then linked via a polypeptide linker sequence to the Fab heavy chain CH1-VH of antibody 2 (Ab2), and the polypeptide linker sequence links the N-terminus of the VH domain of the Fab heavy chain of said Ab1 to the C-terminus of the CH1 domain of said Ab2, and the four light chains comprise the Fab light chain CL-VL of Ab1 and the Fab light chain CL-VL of Ab2 associated with their cognate heavy chain domains; wherein Ab1 and Ab2 recognize different epitopes, and wherein the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain derived from said CH1 domain by substituting the threonine residue at position 192 of the CH1 domain of human IgG1 immunoglobulin with an aspartic acid residue, and the cognate CL domain is a kappa-type mutated domain derived from said CL domain by substituting the asparagine residue at position 137 of the human immunoglobulin CL domain with a lysine residue and substituting the serine residue at position 114 of said CL domain with an alanine residue, and wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutated domain derived from said CH1 domain by substituting the leucine residue at position 124 of the CH1 domain of human IgG1 immunoglobulin with a glutamine residue and substituting the serine residue at position 188 of said CH1 domain with a valine residue, and the cognate CL domain is a kappa-type mutated domain derived from said CL domain by substituting the valine residue at position 133 of the human immunoglobulin CL domain with a threonine residue and substituting the serine residue at position 176 of said CL domain with a valine residue.

5. The protein construct of claim 4, wherein the Fab CH1 domain of Ab1 is a mutated domain derived from said CH1 domain by substituting the threonine residue at position 192 of the CH1 domain of human IgG1 immunoglobulin with an aspartic acid residue, and the cognate CL domain is a kappa-type mutated domain derived from said CL domain by substituting the asparagine residue at position 137 of the human immunoglobulin CL domain with a lysine residue and substituting the serine residue at position 114 of said CL domain with an alanine residue, and wherein the Fab CH1 domain of Ab2 is a mutated domain derived from said CH1 domain by substituting the leucine residue at position 124 of the CH1 domain of human IgG1 immunoglobulin with a glutamine residue and substituting the serine residue at position 188 of said CH1 domain with a valine residue, and the cognate CL domain is a kappa-type mutated domain derived from said CL domain by substituting the valine residue at position 133 of the human immunoglobulin CL domain with a threonine residue and substituting the serine residue at position 176 of said CL domain with a valine residue.

6. The protein construct of claim 4, wherein the Fab CH1 domain of Ab2 is a mutated domain that is derived from the CH1 domain of human IgG1 immunoglobulin by substitution of the threonine residue at position 192 with an asparagine residue, and the homologous CL domain is a kappa-type mutated domain that is derived from the CL domain of human immunoglobulin by substitution of the asparagine residue at position 137 with a lysine residue and substitution of the serine residue at position 114 with an alanine residue, and wherein the Fab CH1 domain of Ab1 is a mutated domain that is derived from the CH1 domain by substitution of the leucine residue at position 124 with a glutamine residue and substitution of the serine residue at position 188 with a valine residue of the CH1 domain of human IgG1 immunoglobulin, and the homologous CL domain is a kappa-type mutated domain that is derived from the CL domain by substitution of the valine residue at position 133 with a threonine residue and substitution of the serine residue at position 176 with a valine residue of the CL domain of human immunoglobulin.

7. The protein construct of claim 1, wherein the polypeptide linker sequence comprises the following amino acid sequence or consists of the following amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX 10 GG (SEQ ID NO:1), wherein X1, X2, X 3、 X4, X5, X6, X7, X8, X9, X 10 are any amino acids that are the same or different.

8. The protein construct of claim 7, wherein the polypeptide linker sequence comprises a sequence selected from or consists of a sequence selected from the following: EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO:2); EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO:3); EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO:4); EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO:5) and EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO:6).

9. The protein construct of claim 1, which recognizes both EGFR and HER2 / neu.

10. The protein construct of claim 9, which is a bispecific antibody, wherein Ab1 and Ab2 are different and are independently selected, on the one hand, from cetuximab or a mutated derivative thereof, or, on the other hand, from trastuzumab or a mutated derivative thereof.

11. The protein construct of claim 1, which recognizes both CD38 and PD-L1.

12. The protein construct of claim 11, which is a bispecific antibody, wherein Ab1 and Ab2 are different and are independently selected, on the one hand, from daratumumab or a mutated derivative thereof, and, on the other hand, from atezolizumab or a mutated derivative thereof.

13. The protein construct of claim 11, which is a bispecific antibody and comprises: a) two heavy chains, each heavy chain consisting of SEQ ID NO: 7; b) four light chains, two of which consist of SEQ ID NO: 15 and the other two consist of SEQ ID NO:

18.

14. A method for producing a protein construct as defined in any one of claims 4 to 13, the method comprising the steps of: a) Culturing a host cell expressing a heavy chain as defined in any one of claims 4 to 13 and a light chain as defined in any one of claims 4 to 13 in a suitable culture medium and under suitable culture conditions; and b) Recovering the produced protein construct from the culture medium or the host cell.

15. The protein construct as defined in any one of claims 9 to 13, which is used as a medicament.

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