RU2779602C2 - Stable multi-specific antibodies - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a protein structure, as well as to its production method. The specified structure contains at least one mutated Fab-fragment.

EFFECT: invention provides for the formation of corresponding pairs of heavy/light chains and prevents the formation of their erroneous pairing.

15 cl, 11 dwg, 6 ex

Description

The invention relates to the production of multispecific antibody molecules that can be used in medicine.

State of the art

Bispecific antibodies (bsAbs) combine the specificity of two antibodies and simultaneously target different antigens or epitopes. BsAbs with "dual-target" functionality can act on a variety of surface receptors or ligands associated with, for example, cancer, proliferation, or inflammation. BsAbs can also identify targets in close proximity, either to promote the formation of a protein complex on a single cell or to facilitate cell-to-cell contacts. Examples of forced binding functionality are bsAbs that promote the formation of protein complexes in the clotting (blood) cascade or are "recruiters" and/or activators of tumor-targeted immune cells. After years of research and development, the first bsAb was approved in 2009.

Initially, bispecific antibodies were generated by chemical coupling or by using quadromas resulting from the fusion of two hybridoma cell lines producing two different mAbs. However, a chemical compound can, in some cases, change the antigen-binding sites, leading to a deterioration in the biological properties of the antibody. The quadroma approach has the disadvantage that random pairing of heavy and light chains from two different antibodies theoretically leads to ten equally possible combinations, which generates a mixture of immunoglobulin molecules, only one of which is the desired bispecific product, which must be separated from the wrong one. paired products.

Currently, the preferred method for obtaining bispecific antibodies is genetic engineering, leading to the creation of a wide variety of different formats of recombinant bispecific antibodies. Some of these bispecific antibodies are very simple and are derived from single chain Fv (scFv) fragments from two (or more) different antibodies linked by a suitable peptide linker. These antibodies are relatively easy to produce, and since they are based on a single polypeptide chain and contain only the Fv regions of the original antibodies, there is no problem of mismatch between chains. However, they are smaller than full-length immunoglobulins and lack constant regions, in particular the Fc region. While their use may be advantageous in some uses, such as when it is desired to avoid Fc-mediated effects, disadvantages occur when Fc-mediated effector function is desired. Also, due to their small size and lack of an Fc region, they have a very short in vivo half-life.

Therefore, other formats of bispecific recombinant antibodies are being developed that more closely mimic the naturally occurring immunoglobulin molecule, and in particular, have an entire Fc region. These formats can be grouped into two main groups.

In the first group of formats (IgG scFv), scFv fragments from antibody A are joined to the ends (mainly C-terminal regions) of the heavy chains of antibody B. The result is an antibody having only one type of heavy chain, which contains VH, CH1, CH2 and CH3 domains of antibody B and VH and VL domains of antibody A, and one type of light chain that contains the VL and CL domains of antibody B, with no mismatch between chains. Such a format is described, for example, by Qu et al. (Blood, 111, 2211-2219, 2008).

In the second set of formats, the heavy chain and light chain from antibody A are paired with the heavy chain and light chain from antibody B. This format reproduces the bispecific antibodies produced by quadromas and hence causes similar mismatch problems. To solve the problem of heavy chain mismatch, it is proposed to modify the CH3 domains of antibodies in order to promote their heterodimerization (ie pairing of heavy chain A with heavy chain B) and prevent their homodimerization. This was done using the so-called "trough-to-trough" approach (Ridgway et al, Protein Eng, 9, 617-21, 1996; US Pat. No. 7,695,936). An overhang mutation of a small amino acid with a larger amino acid is introduced on the CH3 surface of the heavy chain dimer of antibody A, resulting in a steric mismatch that prevents homodimerization. At the same time, in order to promote heterodimerization, a complementary "trough" mutation, consisting in 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 is proposed to use antibodies of different specificity, but sharing a common light chain previously established from a scFv phage library (Merchant et al., Nat Biotechnol, 16, 677-81, 1998; US Pat. No. 7,183,076). The disadvantage of this approach is the difficulty in identifying antibodies that share a common light chain.

International patent application WO2013/005194 suggests that it is possible to prevent heavy chain/light chain mismatch and thus achieve the desired chain alignment by changing some key residues at the interface of the CH1 and CL domains.

Disclosure of invention

In this work, the inventors have found that a new set of mutations can further improve the chain matching in a multispecific antibody molecule.

The invention relates to multispecific, for example, bispecific, antibody-based constructs containing various Fab fragments having a specific set of mutations on the contact surface of the CH1 and CL domains, these mutations promote heavy chain/light chain cognate pairing and prevent their mismatch.

Sequence numbering used in this document for CH1 and CL domains refers to Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n ° 91-3242, pp 662,680,689, 1991).

This document proposes a modified Fab fragment selected from:

a) Fab-fragment, consisting of:

- VH and VL domains of the antibody of interest;

- CH1 domain, which is derived from the CH1 domain of an immunoglobulin by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and

a CL domain which is derived from an immunoglobulin CL domain by replacing the asparagine residue at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue; and

b) Fab-fragment, consisting of:

- VH and VL domains of the antibody of interest;

- CH1 domain, which is derived from the CH1 domain of an immunoglobulin by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue; and

- a CL domain which is derived from an immunoglobulin CL domain by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing the serine residue at position 176 of said CL domain with a valine residue.

Any construct, preferably any protein construct, containing such a reshaped Fab fragment is part of the present invention.

In particular, a multispecific antigen-binding fragment is provided, containing at least two Fab fragments with different CH1 and CL domains, with each Fab fragment recognizing a different epitope of interest, while these Fab fragments are sequentially arranged in any order, wherein the C-terminal region of the CH1 domain of the first Fab fragment is connected to the N-terminal region of the VH domain of the next Fab fragment using a polypeptide linker,

wherein at least one Fab fragment, and more preferably two fragments, is selected from the group consisting of

a) Fab fragment consisting of

- VH and VL domains of the antibody;

- CH1 domain, which is obtained from the CH1 domain of an immunoglobulin by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and

- CL domain, which is obtained from the CL domain of an immunoglobulin by replacing the asparagine residue at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue; and

b) Fab-fragment, consisting of

- VH and VL domains of the antibody;

- CH1 domain, which is obtained from the CH1 domain of an immunoglobulin by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue; and

- CL domain, which is obtained from the CL domain of an immunoglobulin by replacing the valine residue at position 133 of the indicated CL domain with a threonine residue and replacing the serine residue at position 176 of the indicated CL domain with a valine residue.

In a preferred embodiment, the CH1 domain is derived from an IgG immunoglobulin, preferably the IgG1 subtype. The CL domain is preferably a kappa type domain. Preferably, for use in human treatment, the immunoglobulin from which the altered CH1 and altered CL domains are derived is a human immunoglobulin.

The VH and VL domains can be derived from any antibody, natural or engineered, that recognizes the epitope that is desired to be targeted.

The modified Fab fragments of the invention can be used in any multispecific antibody construct where heavy chain/light chain cognate pairing is to be promoted and mismatch prevented.

Preferably, the modified Fab fragments can be used in a multispecific antibody molecule containing antigen binding fragments, each of which consists mainly of consecutive Fab fragments separated by linkers.

Therefore, another object of the invention is a multispecific antigen binding fragment containing at least two and up to six different Fab fragments selected from:

- Fab fragment (also referred to in the description as "wild-type Fab fragment") containing CH1 and CL domains of wild-type immunoglobulin

- mutated Fab fragment (a) as defined above;

- mutated Fab fragment (b) as defined above;

each Fab fragment recognizes a different epitope of interest, and these Fab fragments are sequential in any order, the C-terminal regions of the CH1 domain of the first Fab fragment are connected to the N-terminal region of the VH domain of the next Fab fragment via a peptide linker;

or one or two Fab fragments are sequential in any order and connected via a peptide linker to the C-terminus of the CH3 domain or the C-terminus of the hinge sequence, while another Fab or two Fabs, consecutively in any order, are connected to the N- end of the hinge sequence.

The above hinge sequence is in most cases a natural hinge sequence derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD, and most preferably from IgG1, or may be a modified sequence through the use of point mutations and/or additions of amino acids in this way that the modified hinge sequence is less than 80 amino acids, preferably less than 60 amino acids, even more preferably less than 40 amino acids.

An "antigen-binding fragment" is defined herein as a molecule having two or more antigen-binding sites, each of which recognizes a different epitope. Different epitopes may be generated by the same antigenic molecule or by different antigenic molecules.

In a preferred embodiment, a multispecific antibody is also provided having two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment, i.e. an antigen-binding fragment capable of binding multiple (ie, more than one) antigens, as defined above.

In a separate embodiment, the multispecific antibody has an immunoglobulin-like structure and contains

- two identical antigen-binding arms, each of which consists of such multispecific antigen-binding fragments as described above;

- dimerized CH2 and CH3 immunoglobulin domains;

- an IgA, IgG or IgD hinge region linking the C-terminal regions of the CH1 domains of the antigen-binding arms to the N-terminal regions of the CH2 domains.

More specifically, the subject of the invention is a multispecific, preferably bispecific, antibody containing two heavy chains and four light chains, with each heavy chain containing

- Fc-region of immunoglobulin containing hinge-CH2-CH3 domains,

- while the Fc region is connected to the Fab of the heavy chain CH1-VH of antibody 1 (Ab1) using the specified hinge domain,

- which, in turn, binds to the heavy chain Fab CH1-VH of antibody 2 (Ab2) using a polypeptide linker sequence, and the polypeptide linker sequence connects the N-terminus of said Ab1 heavy chain VH domain Fab to the C-terminus of said Ab2 domain CH1,

and four light chains contain CL-VL Ab1 light chain Fabs and CL-VL Ab2 light chain Fabs linked to their heavy chain cognate domains;

while Ab1 and Ab2 recognize different epitopes,

and wherein the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain that is derived from an immunoglobulin CH1 domain by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and the related CL domain is a mutated domain that is derived from an immunoglobulin CL domain by replacing the asparagine residue at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue, and/or

wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutated domain that is derived from an immunoglobulin CH1 domain by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue, and related The CL domain is a mutated domain that is derived from the CL domain of an immunoglobulin by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing the serine residue at position 176 of said CL domain with a valine residue.

Any protein chain selected from:

the light chain of the mutated Fab fragment of the invention;

- the heavy chain of the mutated Fab fragment of the invention;

- heavy chain antigennegative fragment of the invention;

- the heavy chain of an immunoglobulin-like multispecific antibody of the invention.

The disclosure further provides a polynucleotide comprising a sequence encoding a protein chain of the invention. The specified polynucleotide may also contain additional sequences, in particular, it may preferably contain a sequence encoding a leader sequence or a signal peptide that provides the secretion of the specified protein chain.

Figure captions

Fig. 1 shows BiXAb-6567 SDS polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Lane 1: movement of BiXAb-6567 under reducing conditions; lane 2: molecular weight markers with the indicated weight of each lane; lane 3: movement of BiXAb-6567 under non-reducing conditions.

Fig. 2 shows the analysis of BiXAb-6567 using size exclusion chromatography.

Fig. 3 shows the melting profiles of the two parent antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 determined by differential scanning calorimetry.

Fig. 4A shows the binding profiles of the two parent antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 in a direct binding ELISA of the CD38 antigen. Fig. 4B shows the binding profiles of the two parent antibodies (anti-CD38 and anti-PD-L1) and BiXAb-6567 in a PD-L1 antigen direct binding ELISA. Fig. 4C shows the binding profiles of BiXAb-6567 in a dual antigen binding ELISA (PD-L1 and CD38).

Fig. 5A - 5C show fluorescence-activated cell sorting profiles of two parent mAbs (anti-CD38 and anti-PD-L1) and BiXAb-6567 on three different cell lines, 5A: multiple myeloma RPMI-8226, 5B: CHO cells stably transfected with full length CD38, and 5C: ovarian cancer cell line SKOV-3.

Fig. 6 shows binding titration profiles on the CHO-CD38 cell line of two parent antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567 and a negative control anti-CD20 antibody.

Fig. 7 shows the cytotoxic activity profiles of two parent antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567, and two negative control antibodies, anti-CD20 and anti-HER2, in ADCC analysis using a multiple myeloma cell line, RPMI-8226 , as target cells and non-fractionated non-activated pre-mononuclear cells as effector cells.

Fig. 8 shows the cytotoxic activity profiles of two parent antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567, and two negative control antibodies, anti-CD20 and anti-HER2, in ADCC analysis using the CHO-CD38 cell line as cells. -targets and unfractionated pre-activated mononuclear cells as effector cells.

Fig. 9 shows the cytotoxic activity profiles of two parent antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567, and two negative control antibodies, anti-CD20 and anti-HER2, in ADCC analysis using the SKOV-3 cell line as cells. -targets and enriched pre-IL-12-activated NK cells as effector cells.

Fig. 10 shows the cytotoxic activity profiles of two parent antibodies (anti-CD38 and anti-PD-L1), BiXAb-6567, and two negative control antibodies, anti-CD20 and anti-HER2, in ADCC analysis using the SKOV-3 cell line as cells. -targets and enriched pre-IL-15-activated NK cells as effector cells.

Fig. 11 is a schematic representation of a bispecific antibody of the invention containing two heavy chains and four light chains, showing different combinations of mutations.

Detailed description of the invention

Definitions:

The basic structure (framework) of a natural antibody molecule is a Y-shaped tetrameric quaternary structure, consisting of two identical heavy chains and two identical light chains, which are held together by non-covalent interactions and by disulfide bonds between the chains.

In different mammalian species, there are five types of heavy chains: α, δ, ε, γ and μ, which define the class (isotype) of immunoglobulin: IgA, IgD, IgE, IgG and IgM, respectively. The N-terminal variable domain (VH) of the heavy chain is followed by a constant region containing three domains (numbered CH1, CH2, and CH3 from N-terminus to C-terminus) in heavy chains γ, α, and δ, while the heavy chain constant region µ and ε consists of four domains (numbered CH1, CH2, CH3 and CH4 from N-terminus to C-terminus). The CH1 and CH2 domains of IgA, IgG and IgD are separated by a flexible hinge region, the length of which varies between different classes and in the case of IgA and lgG between different isotypes: lgG1, lgG2, IgG3 and IgG4 have hinges of 15, 12, 62 (or 77) respectively , and 12 amino acids, and IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

There are two types of light chains, λ and κ, which can bind to any heavy chain isotype, however, in a given antibody molecule, both chains are chains of the same isotype. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a single domain constant region called CL.

The heavy and light chains are paired by protein-protein interactions between the CH1 and CL domains and by VH/VL interactions, and the two heavy chains are paired by protein-protein interactions between their CH3 domains. The structure of the immunoglobulin molecule is in most cases stabilized by interchain disulfide bonds between the CH1 and CL domains and between the hinges.

The antigen binding sites correspond to the arms of a Y-shaped structure, each of which consists of a full light chain paired with the VH and CH1 domains of the heavy chain, and are called Fab fragments (antigen binding fragments). Fab fragments were first generated from native immunoglobulin molecules by papain digestion, which cleaves the antibody molecule at the hinge region on the amino-terminal side of the interchain disulfide bonds, thus releasing two identical antigen-binding arms. Other proteases, such as pepsin, also cleave the antibody molecule at the hinge region, but at the carboxy-terminal side of the interchain disulfide bonds, releasing fragments consisting of two identical Fab fragments and remaining linked via disulfide bonds; reduction of disulfide bonds in F(ab')2 fragments gives Fab' fragments.

The part of the antigen-binding site corresponding to the VH and VL domains is called the Fv fragment (variable fragment); it contains CDRs (complementarity determining regions) that form the antibody's antigen-binding site (also called the paratope).

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

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

In the context of the invention, a "multispecific antigen binding fragment" is defined as a molecule having two or more antigen binding sites, each of which recognizes a different epitope. Different epitopes may be generated by the same antigenic molecule or by different antigenic molecules. The term "recognition" or "recognises" means that the fragment specifically binds to the target antigen.

A “multispecific antibody” consists of at least two multispecific antigen binding fragments/arms and is capable of binding two, three or more different antigens.

An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily and/or more sustainably than it binds to other substances. “Specific binding” or “preferred binding” does not necessarily require (although it may be included) exclusive binding. As a rule, but not necessarily, the reference to binding means the preferred binding.

The term “mutated derivative”, “mutant”, or “functional variant” means a sequence that differs from the original sequence to which it is related by a deletion, substitution, or insertion of one or more amino acids. Preferably the mutated derivative preferably shows at least 80%, preferably at least 85%, again preferably at least 90% sequence homology with the native sequence. In a particular embodiment, the mutations do not significantly affect the function of the antibody.

Mutated derivatives or functional variants may contain a VH chain that contains at least 85% amino acid sequence (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any of the reference sequences described herein, a VL chain that has an amino acid sequence of at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98 % or 99%) identical to either or both of the reference sequences described in this document. These variants are capable of binding to target antigens. In some examples, the variants have similar antigen binding affinities to the reference antibodies described above (eg, have a KD of less than 1 x 10 ^-7 M, 10 ^-8 M, preferably less than 1 x 10 ^-9 or 1 x 10 ^-10 M).

Binding affinity is defined in terms of ka (association rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). Generally, specific binding, when used in relation to an antibody, refers to an antibody that specifically binds (“recognizes”) its target(s) with an affinity value (KD) of less than 10 ^-7 M, preferably less than 10 ^-8 M, for example, less than 10 ^-9 M or 10 ^-10 M. A lower KD value indicates a higher binding affinity (i.e., stronger binding), such that a KD value of 10 ^-9 indicates a higher binding affinity than a KD value 10 ^-8 .

The “percent identity” of two amino acid sequences is determined using the Karlin and Altschul Proc algorithm. Natl. Acad. sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. sci. USA 90:5873-77, 1993. Such an algorithm is part of the NBLAST and XBLAST programs (version 2.0) Altschul, et al. J. Mol. Biol. 215:403-10, 1990. A BLAST protein search can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of interest. In the event that there are gaps between two sequences, Gapped BLAST can be used 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 (eg, XBLAST and NBLAST) can be used.

In other embodiments, the functional variants described herein may contain one or more mutations (eg, conservative substitutions) that preferably do not occur in residues that are putatively interacting with one or more of the CDRs.

This document describes mutated derivatives or functional variants that are substantially identical to a reference antibody.

The term “substantially identical (substantially identical)” or “substantially (different)” means that the respective amino acid sequences (e.g., in the framework regions (FRs), CDRs, VH, or VL domain) of the variant differ insignificantly (e.g., including conservative amino acid substitutions ) compared to the reference antibody such that the variant has substantially similar binding activity (eg, affinity, specificity, or both) and bioactivity relative to the reference antibody.

Such a variant may include minimal amino acid changes, such as 1 or 2 substitutions in the 5 amino acid sequence of a particular region. Generally, more substitutions can be made in the FR regions, as opposed to the CDR regions, as long as they do not adversely affect the binding function of the antibody (eg, more than 50% reduction in binding affinity compared to the parent antibody). In some embodiment, the sequence identity may be about 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater between the original and the modified antibody. In some embodiments, the modified antibody has the same binding specificity and has an affinity that is at least 50% of that of the original antibody.

Conservative substitutions will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are derived. For example, a "conservative amino acid substitution" may include 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) are determined by those skilled in the art. For example, amino acid substitutions can be used to identify important residues in the sequence of a molecule, 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 of modifying the polypeptide sequence known to those of ordinary skill in the art, for example, such methods can be found in the following references, for example, 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 amino acid substitutions include substitutions made among 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.

Amino acid sequence variants of an antibody can be generated 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 sequences of the antibody. Any combination of deletion, insertion, and substitution is done in order to obtain the final construct, provided that the final construct has the desired characteristics. Nucleic acid molecules encoding variants of the amino acid sequences of an antibody can be obtained using a number 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 a previously generated variant or non-variant (natural) version of an antibody. In one embodiment, the value of the equilibrium dissociation constant (KD) of the antibodies of the invention is less than 10 ^-7 M, in particular less than 10 ^-8 M, 10 ^-9 M or 10 ^-10 M. Binding affinity can be determined using known in this field technique methods such as ELISA or bispecific interaction analysis (eg using surface plasmon resonance), or other methods known in the art.

Any of the molecules described in this document can be investigated to determine its properties, such as antigen-binding activity, antigen-binding specificity, and biological functions using standard methods.

The terms "subject", "individual" and "patient" are used interchangeably herein and refer to the mammal being evaluated for treatment and/or being treated. The subject may be a human, but other mammals are also included, in particular mammals useful as laboratory models of human disease, eg mouse, rat, rabbit, dog, etc.

The term "treatment" refers to the action, use or therapy in which a subject, including a human being, is subjected to medical treatment in order to improve the condition of the subject, directly or indirectly. In particular, the term refers to the reduction in the number of cases or alleviation of symptoms, the elimination of relapses, the prevention of relapses, the prevention of morbidity, the improvement of symptoms, the improvement of prognosis, or a combination of these in some embodiments. The skilled artisan will appreciate that treatment does not necessarily result in complete absence or elimination of symptoms. For example, in relation to cancer, "treatment" may refer to reducing the growth, proliferation or metastasis of neoplastic or malignant cells, preventing or delaying the growth, proliferation or metastasis of neoplastic or malignant cells, or any combination of these processes.

Design of preferred multispecific antibodies:

Provided herein are multispecific antigen binding fragment(s) and multispecific antibody constructs containing said fragments, with each multispecific antigen binding fragment consisting primarily of consecutive Fab fragments separated by a linker of the invention.

Such fragments and constructs preferably contain chains from human immunoglobulins, preferably IgG, again preferably IgG1.

In case the multispecific antigen binding fragment contains more than two different Fab fragments, the polypeptide linkers separating the Fab fragments may be identical or different.

According to a preferred embodiment of the multispecific antibody of the invention, it has two identical antigen-binding arms, each consisting of antigen-binding fragments capable of binding a number (more than one) of antigens, as described above. Antigen-binding arms can be connected together in different ways, depending on the intended use of the antibody.

If it is desired to produce an antibody without Fc-mediated effects, the antibody should not contain an Fc region. In this case, the two antigen-binding arms can be linked together, for example:

- by homodimerizing the antigen-binding arms via interchain disulfide bonds provided by the polypeptide linker(s) separating Fab fragments, if said linker(s) contains cysteine residues; and/or

- by adding at the C-terminal region of each antigen-binding arm of a polypeptide extension containing cysteine residues, allowing the formation of interchain disulfide bonds, and homodimerizing said polypeptide extension, which generates a hinge-like structure; by way of non-limiting examples, the specified polypeptide extension may be, for example, the hinge sequence of IgG1, IgG2, IgG3, IgA or IgD;

- by means of a semi-rigid linker connecting the C-terminal regions of the heavy chains of the two antigen-binding arms in order to form one polypeptide chain and keep said antigen-binding arms at a sufficient distance from each other.

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

In this case, the multispecific antibody of the invention has an immunoglobulin-like structure comprising:

two identical multispecific antigen-binding arms as described above;

- dimerized CH2 and CH3 immunoglobulin domains;

- any IgA, IgG or IgD hinge region connecting the C-terminal regions of the CH1 domains of the antigen-binding arms to the N-terminal regions of the CH2 domains, or alternatively, in the case that the CH4 domains that follow the CH3 domains are derived from IgM or IgE, The C-terminal regions of the CH1 domains of the antigen-binding arms can bind directly to the N-terminal regions of the CH2 domains.

Preferably, the CH2 and CH3 domains, the hinge region and/or the CH4 domains are derived from the same immunoglobulin, or from immunoglobulins of the same isotype and subclass, as the CH1 domains of the antigen binding arm.

Can be used CH2, CH3 and optionally CH4 domains, as well as hinge regions of native immunoglobulins. You can also modify (mutate) them, if desired, for example, in order to modulate the effector function of the antibody. In some cases, all or part of the CH2 or CH3 domain may be missing.

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

Such preferred antibodies of the invention are full length antibodies. They preferably contain heavy chains and light chains from human immunoglobulins, preferably IgG, still preferably IgG1.

The light chains may be lambda or kappa light chains, preferably they are kappa light chains.

In a preferred embodiment, the polypeptide linker used in the invention links two pairs of IgG Fab domains into a tetra-Fab format of a multispecific, preferably bispecific antibody, the amino acid sequence of which contains the heavy chain sequences of at least two Fab linked by a peptide linker followed by a hinge sequence followed by an IgG Fc sequence co-expressed with the corresponding IgG light chain sequences.

An example of preferred bispecific antibodies of the invention that have an IgG-like structure is shown in FIG. eleven.

In a preferred embodiment, the bispecific antibodies of the invention typically comprise

- a continuous heavy chain constructed from Fc (hinge-CH2-CH3),

- followed by the Fab of the heavy chain of antibody 1 (CH1-VH) and the subsequent Fab of the heavy chain (CH1-VH) of antibody 2, the latter connected by a polypeptide linker sequence derived from the hinge region,

- and during protein expression, the resulting heavy chain assembles into dimers, while the co-expressed light chains (VL-CL) of antibody 1 and antibody 2 combine with their cognate heavy chains to form the final tandem F(ab)'2- Fc molecule,

antibody 1 (Ab1) and antibody 2 (Ab2) are different.

In a preferred embodiment, bispecific antibodies are described containing

- two Fab fragments with different mutated CH1 and mutated CL domains, consisting of

a) Fab fragment having mutated CH1 and mutated C-kappa domains derived from human IgG1/kappa and VH and VL domains of Ab1,

b) a Fab fragment having mutated CH1 and mutated C-kappa domains derived from human IgG1/kappa and VH and VL domains of Ab2,

c) a mutated light chain constant domain that is derived from the human kappa constant domain,

Fab fragments are sequentially arranged in the following order

- the C-terminal region of the mutated CH1 domain of the Fab fragment of Ab1 is associated with the N-terminal region of the VH domain of the Fab fragment of Ab2 via a peptide linker,

- the human IgG1 hinge connects the C-terminal regions of the mutated CH1 domain of the Ab2 fragment to the N-terminus of the CH2 domain,

- dimerized CH2 and CH3 domains of human IgG1.

Ab1 and Ab2 can be any antibody of interest, in particular any antibody of interest in therapeutic medicine.

In a separate embodiment, Ab1 and Ab2, while different, are independently selected from the group consisting of an anti-CD38 antibody (such as daratumumab) and an anti-PD-L1 antibody (such as atezolizumab).

In another specific embodiment, Ab1 and Ab2, being different, are independently selected from the group consisting of an anti-EGFR antibody and an anti-HER2/neu receptor. In a preferred embodiment, Ab1 and Ab2, being different, are independently selected from the group consisting of cetuximab or a modified derivative thereof, on the one hand, and trastuzumab, or a modified derivative thereof, on the other hand.

Such antibodies are useful as a medicament, more particularly in the treatment of cancer.

In a specific example, the bispecific molecule is a bispecific anti-CD38, anti-PD-L1 antibody that contains, preferably consists of a) two heavy chains, each of which contains, preferably consists of SEQ ID NO: 7 and b) four light chains, two of which contain, preferably consist of SEQ ID NO:15, and the other two contain, preferably consist of SEQ ID NO: 18. Such a bispecific antibody is designated BiXAb-6567.

SEQ ID NO:7 (heavy chain) is:

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPQAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVDVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This heavy chain sequence contains

- VH daratumumab (SEQ ID NO:8)

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

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

ASTKGPSVFPQAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLVSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

- AP linker (SEQ ID NO:3)

EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG

- VH of atezolizumab (SEQ ID NO: 10)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

- CH1 domain (human IgG1 G1m(3) allotype with T192D Fab mutations of atezolizumab (SEQ ID NO:11)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVDVPSSSLGTQTYICNVNHKPSNTKVDKRV

- Hinge region of human IgG1 (SEQ ID NO:12)

EPKSCDKTHTCPPCP

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

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

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

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain SEQ ID NO: 15 (daratumumab) is

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLVSTLTLSKADYEKHKVYACESFTHQGLSSPV

This light chain sequence contains

- VL daratumumab (SEQ ID NO:16)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

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

RTVAAPSVFIFPPSDEQLKSGTASVTCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLVSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Light chain SEQ ID NO: 18 (atezolizumab) is

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSYVACEVTHQ

This light chain sequence contains

- VL atezolizumab (SEQ ID NO:19)

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK

- Cappa domain of atezolizumab with mutations S114A and N137K (SEQ ID NO:20)

RTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The third pair of Fab domains (directed against different target antigens than the first and second pair of Fab domains) can also be linked to an identical or different polypeptide linker: i) or through the C-terminus of the heavy chains of said third pair of Fab domains with the N-terminus of the heavy chains of outer (ie, Fc-distal) Fab domains, ii) or through the C- or N-terminus of the heavy chains of said third pair of Fab domains with the C-terminus of both heavy chains of the Fc domain.

Any of the molecules described herein may be modified to contain additional non-protein moieties that are known in the art and readily available, such as by pegylation or hyperglycosylation. Modifications that may increase serum half-life or resistance to proteolytic cleavage are also contemplated.

The antibodies of the invention may or may not be glycosylated, or may exhibit a range of glycosylation profiles. In a preferred embodiment, the antibodies are non-glycosylated in the heavy chain variable region but are glycosylated in the Fc region.

Humanized forms of the original non-human antibody may be used. In the humanization approach, complementarity determining regions (CDRs) and some other amino acids from donor variable regions are "grafted" into human variable acceptor regions and then fused to human constant regions. See, for example, Riechmann et al., Nature 332:323-327 (1988); U.S. Patent No. 5,225,539.

Another example of constructs of the invention is a Fab-Fab bispecific antigen binding fragment that does not contain an Fc domain.

Such Fab-Fab constructs typically contain two different Fab domains. Such antibodies have only one Fab domain that binds to antigen 1 and antigen 2. They have the same light chains as in the corresponding BiXAb antibodies; however, the Fab-Fabs heavy chains are shortened such that their C-terminalmost residue is cysteine-220 (in EU numbering).

Another example of constructs of the invention is a dimerized Fab-Fab construct (eg, either via a disulfide in the linker or via natural hinge disulfides at the C terminus of the C-terminal-proximal Fab domain).

Linker design

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

Optionally, said polypeptide linker may further comprise the sequence of 2 to 15, preferably 5 to 10 N-terminal amino acids of the CH2 domain of the immunoglobulin.

The polypeptide linker sequence is in most cases less than 80 amino acids, preferably less than 60 amino acids, still preferably less than 40 amino acids.

In some cases, sequences from native hinge regions may be used; in other cases, point mutations may be introduced into these sequences, such as substitution of one or more cysteine residues in native IgG1, IgG2, or IgG3 hinge regions for alanine or serine, in order to avoid undesired intra- or inter-chain disulfide bonds.

In a separate embodiment, the polypeptide linker sequence comprises or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO:1) wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ , X ₉ , X10, identical or different, are any amino acid. In particular, the sequence of the polypeptide linker may contain or consist of a sequence selected from the group consisting of

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).

In a separate embodiment, X ₁ , X ₂ and X ₃ , the same or different, are threonine (T) or serine (S).

In another specific embodiment, X ₁ , X ₂ and X3, the same or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile ( I), preferably X ₁ , X2 and X ₃ , the same or different, may be Ala (A) or Gly (G).

Alternatively, X ₁ , X ₂ and X ₃ , the same or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe ( F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

In a particular embodiment, X ₄ and X ₅ , the same or different, are any amino acid selected from the group consisting of serine (S), cysteine (C), alanine (A), and glycine (G).

In a preferred embodiment, X ₄ is serine (S) or cysteine (C). In a preferred aspect, X ₅ is alanine (A) or cysteine (C).

In a separate embodiment, X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , the same or different, are any amino acid other than threonine (T) or serine (S). Preferably X ₆ , X ₇ , X ₈ , X ₉ , X10, identical or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I).

Alternatively, X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , the same or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

In a preferred embodiment, X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , the same or different, are selected from the group consisting of Ala (A) and Gly (G).

In yet another preferred embodiment, X6 and X7 are the same and are preferably selected from the group consisting of Ala (A) and Gly (G).

In a preferred embodiment, the polypeptide linker sequence contains or consists of the sequence of SEQ ID NO: 1, in which

X ₁ , X ₂ and X ₃ , identical or different, are threonine (T), serine (S);

X ₄ is serine (S) or cysteine (C);

X ₅ is alanine (A) or cysteine (C);

X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , identical or different, are selected from the group consisting of Ala (A) and Gly (G).

In another preferred embodiment, the polypeptide linker sequence contains or consists of the sequence of SEQ ID NO: 1, in which

X ₁ , X ₂ and X ₃ , the same or different, are Ala (A) or Gly (G);

X ₄ is serine (S) or cysteine (C);

X ₅ is alanine (A) or cysteine (C);

X ₆ , X ₇ , X ₈ , X ₉ , X ₁₀ , identical or different, are selected from the group consisting of Ala (A) and Gly (G).

In case the multispecific antigen binding fragment contains more than two different Fab fragments, the polypeptide linkers separating the Fab fragments may be the same or different.

Obtaining multispecific antibodies:

The skilled artisan may refer to International Patent Application WO2013/005194, incorporated herein by reference, for general technical methods for expressing multispecific antibodies.

In addition, this document describes a polynucleotide containing a sequence encoding the protein chain of a molecule or antibody of the invention. The specified polynucleotide may also contain additional sequences: in particular, it may preferably contain a sequence encoding a leader sequence or a signal peptide that provides the secretion of the specified protein chain. Also disclosed are host cells transformed with said polynucleotide.

In most cases, the amino acid sequences of different monoclonal antibodies are used to generate DNA sequences, optionally after codon optimization for mammalian expression. For the heavy chain, DNA encoding signal peptides, a Fab1 variable region and a CH1 constant domain followed by a hinge linker and a variable region, and a Fab2 constant CH1 domain with flanking sequences are synthesized for restriction enzyme cleavage. For the light chain, DNA encoding signal peptides and variable and constant kappa regions is synthesized.

Nucleic acids encoding the heavy and light chains of the antibodies of the invention are inserted into expression vectors. Light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector(s) that allow expression of the immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. In most cases, expression vectors replicate in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors contain selectable markers, such as tetracycline or neomycin, to enable the detection of cells transformed with the desired DNA sequences.

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

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

Specifically, the three vectors may preferably be used in the following molecular ratio of 2:1:1 (HC:LC1:LC2).

A host cell transfected with an expression vector containing a polynucleotide that encodes 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 site of the specified heavy chain; a second light chain that specifically pairs with a second VH/CH1 region of said heavy chain.

In a further embodiment, the host cell may be further transformed with a polynucleotide encoding a third light chain that is different from the first and second light chains and that specifically pairs with the third VH/CH1 region of said heavy chain.

The recombinant vectors for expressing the antibodies described herein typically contain a nucleic acid encoding antibody amino acid sequences operably linked to a promoter, either constitutive or inducible. Vectors may be suitable for replication and integration into prokaryotic, eukaryotic, or both. Exemplary vectors contain transcription and translation terminators, initiation sequences, and promoters suitable for regulating the expression of a nucleic acid encoding an antibody. The vectors optionally contain common expression cassettes containing at least one independent terminator sequence, sequences that allow the cassette to replicate in both prokaryotic and eukaryotic cells, i.e. shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

The multispecific, eg, bispecific antibodies described herein can be produced in a prokaryotic or eukaryotic expression system such as bacteria, yeast, filamentous fungi, plant cells, insects (eg using a baculovirus vector) and mammalian cells. Optionally, the recombinant antibodies of the invention are glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of suitable mammalian host cell lines are human embryonic kidney cells (293 cells), newborn hamster kidney cells (BHK cells), Chinese hamster ovary cells /− or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells) and human liver cells (Hep G2 cells).

Mammalian tissue cell cultures are preferred for the expression and production of polypeptides because a large number of suitable host cell lines capable of secreting intact immunoglobulins have been established in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines ( such as NS0) or transformed B cells or hybridomas.

In a most preferred embodiment, the multispecific, eg, bispecific, antibodies of the invention are produced using a CHO cell line, most preferably a CHO-S or CHO-DG-44 cell line, or derivatives thereof.

Expression vectors for these cells may include expression control sequences such as an origin of replication, a promoter, an enhancer, and information processing sites such as a ribosome binding site, RNA splicing sites, polyadenylation sites, and a transcription termination sequence. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, and the like.

Vectors containing polynucleotide sequences of interest (eg, heavy and light chain coding sequences and expression control sequences) can be transferred into the host cell using well known methods, varying depending on the host cell type. For example, calcium phosphate treatment or electroporation can be used on different host cells. (See Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). When heavy and light chains are cloned into different expression vectors to obtain expression and assembly of intact immunoglobulins, the vectors are transfected together.

Host cells are transformed or transfected with vectors (eg, by chemical transfection or electroporation) and cultured in standard nutrient media (or suitably modified) to induce promoters, select transformants, or amplify genes encoding the desired sequences.

Expression of antibodies can be transient (transient) or stable.

Preferably, multispecific, for example, bispecific antibodies are produced by methods that provide stable expression, while cell lines stably transfected with DNA encoding all polypeptide chains of a multispecific, for example, bispecific antibody, such as BiXAb-6567, are capable of long-term expression, allowing the production of therapeutic agents. For example, stable expression in the CHO cell line is particularly effective.

Once the complete antibodies, their dimers, single light and heavy chains, or other forms of the immunoglobulins of the present invention have been expressed, they can be isolated or purified to provide substantially homogeneous preparations for further analysis and use. Standard protein purification methods well known in the art can be used. For example, suitable purification methods may include immunoaffinity or ion exchange column fractionation, ethanol precipitation, high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate fractionation, and gel filtration (see Scopes, Protein Purification (Springer-Verlag, N.Y., 1982) For pharmaceutical use, substantially pure immunoglobulins that are at least about 90-95% homogeneous are preferred, and immunoglobulins with 98-99% homogeneity are most preferred.

In vitro production allows scaling up to produce large amounts of the desired multispecific, eg, bispecific antibodies of the invention. Such methods may use a homogeneous suspension culture, eg in an airlift or continuous mixing reactor, or immobilized or encapsulated cell culture, eg in hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges.

Application

Protein constructs or multispecific antibodies of the invention can be used in all areas of application of multispecific antibodies. In particular, they can be used to obtain drugs suitable for use in a wide range of therapeutic and diagnostic methods in patients (conjugated with radioactive, fluorescent, chemiluminescent or any other labels), or in vitro, (for example, for staining cells or tissues when used methods of immunohistochemistry, immunoblotting and immunofluorescence). These medical and diagnostic products are also part of the subject matter of the invention.

Another aspect of the invention is a pharmaceutical composition containing a protein construct or antibody according to the invention. Another aspect of the invention is the use of a protein construct or antibody of the invention for the manufacture of a pharmaceutical composition. Another aspect of the invention is a method for the manufacture of a pharmaceutical composition containing a protein construct or antibody according to the invention.

In another aspect, the present invention provides a composition, eg, a pharmaceutical composition, comprising a protein construct or antibody as defined herein, formulated with a pharmaceutical carrier.

The composition of the present invention can be administered using a variety of methods known in the art.

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

Examples

Example 1 Preparation of the bispecific antibody of the invention BiXAb-6567

Gene synthesis

Anti-CD38 (daratumumab) and anti-PDL1 (atezolizumab) amino acid sequences were used to generate DNA sequences, after codon optimization for mammalian expression using the GeneScript program. These antibodies are referred to as “parent” anti-CD38 and “parent” anti-PD-L1 mAbs.

The heavy chain DNA construct was designed as a signal peptide followed by the sequence SEQ ID NO:7 [consisting of the variable region followed by the Fab1 (anti-CD38) constant CH1 domain mutated with Leu mutations on Gln and Ser to Val according to Kabat at positions 124 and 188 according to Kabat, respectively, followed by a linker and then a variable region, followed by a Fab2 constant CH1 domain (anti-PD-L1) mutated with Thr to Asp at position 192 according to Kabat]; flanking sequences for restriction enzyme digestion were introduced at both ends of the heavy chain DNA construct. The DNA construct for the light chain was designed as a signal peptide (SEQ ID NO:21) followed by a variable region and then a constant kappa region. For anti-CD38 light chain (SEQ ID NO:15), mutations were introduced at positions 143 (Leu to Gln) and 188 (Ser to Val) according to Kabat in the constant kappa domain. For anti-PDL1 light chain (SEQ ID NO:18), mutations at positions 133 (Val to Thr) and 176 (Ser to Val) according to Kabat were introduced into the constant kappa domain. All DNA constructs were synthesized by Gene Art.

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

Expression and purification

Bispecific antibody BiXAb-6567 was generated using transient gene expression by cotransfection of 3 genes encoded on separate vectors in a molecular ratio of 2:1:1 = HC:LC1:LC2 (1 continuous heavy chain (HC) and 2 light chains (LC) ) in serum-adapted CHO-S cells in suspension (CHO SFM-II medium, Life Technologies™). Typically, for 50 ml scale expression, a total of 50 µg of 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 , then 1 ml of CHO SFM medium containing 25 µl of 3 mg/ml PEI transfection reagent pH7.0 (Polyplus) was added, then the reaction mixture was incubated at room temperature for 20 minutes. The DNA-PEI mixture was then added to 49 ml CHO-S cells (Life Technologies' Invitrogen FreeStyle™) at 1~2x 10 ⁶ /ml in a 125 ml shake flask. Cells were shaken for 6 days. The supernatant was collected by centrifugation at 3000 rpm for 15 minutes. The expression titer of BiXAb-6567 in the supernatant was determined using ForteBio protein A biosensors (Octet® Systems). BiXAb-6567 was then purified by protein A resin affinity chromatography (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 filters, Techno Plastic Products AG) and the final concentration determined by measuring the optical density (OD) at 280 nm (Eppendorf BioSpectrometer®).

In most cases, BiXAab-6567 showed a good expression titer (> 180 mg/liter) with transient CHO expression. This level of expression is comparable to the level of expression seen with conventional monoclonal antibodies.

Polyacrylamide gel electrophoresis in the presence of SDS

To assess the quality of the purified BiXAb-6567, we performed SDS-PAGE (Experion™ automated electrophoretic system, BioRad). In the presence of sodium dodecyl sulfate (SDS) in the running buffer, the rate at which the antibody moves in the gel depends mainly on its size, which makes it possible to determine the molecular weight. This analysis was carried out under non-reducing conditions and under reducing conditions; the latter allow the destruction of disulfide bonds and hence the visualization of individual polypeptide chains (light chains and heavy chain).

The SDS-PAGE results are shown in FIG. 1. Under non-reducing conditions, the quaternary structure of the antibody is maintained and the observed molecular weight should be the sum of the molecular weights of the different heavy and light chains. The bispecific antibody of the 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, if post-translational modifications (PTM) are ignored, such as N-glycosylation in Fc on asparagine 297. The gel was calibrated with a mixture of known molecular weight standards. Under non-reducing conditions, the result is a major band almost equal to the standard at 250 kDa molecular weight, consistent with the calculated molecular weight and predicted glycosylation of the two asparagines at position 297 in the Fc domain. Under reducing conditions, dithiothreitol (DTT) further denatures BiXAb-6567 by reducing disulfide bonds and destroying the quaternary structure, and thus the six polypeptide chains must be mixed separately in the gel according to their molecular weight. Two identical heavy chains of BiXAb-6567 co-move as a single lane, and two pairs of light chains, due to their almost identical molecular weight, co-move as a second lane. Therefore, the results showed two major bands, approximately 75 kDa and 25 kDa, based on the mobility of known molecular weight standards. Each heavy chain has one N-glycosylation site on asparagine 297 which accounts for the higher molecular weight band width, with the observed molecular weight slightly higher than calculated (75.44 kDa); this increase is typical of glycosylated proteins. The calculated molecular weights of the light chains of anti-CD38 (23.40 kDa) and anti-PD-L1 (23.36 kDa) are very close, which leads to their co-movement.

In conclusion, BiXAb-6567 SDS-PAGE showed the expected profiles under both non-reducing and reducing conditions and was consistent with the calculated theoretical molecular weights, taking into account the existence of an N-glycosylation site in the heavy chain.

SEC Analysis

Protein aggregation is often observed in engineered protein molecules. We performed Analytical Size Exclusion Chromatography (SEC) analysis to quantify the high molecular weight content of a one-step affinity-purified BiXAb-6567 preparation (see Expression and Purification of Variants). We used a SEC-s3000 (300x 7.8 mm) column (BioSep) and an Aktapurifier 10 system (GE Healthcare); analysis was performed at a flow rate of 1 ml/min using PBS buffer pH 7.4.

The SEC chromatogram shown in Fig. 2 showed that the main peak corresponded to the expected size of monomeric BiXAb-6567; this peak represented 98.2% of the total sample. In addition, a small peak was observed corresponding to higher molecular weight molecules (possibly dimers); this peak represented 1.8% of the total sample. Thus, we were able to conclude that the percentage of higher molecular weight molecules is negligible and similar to conventional monoclonal antibodies produced in CHO expression systems. The narrow and symmetrical shape of the monomeric peak suggests that BiXAb-6567 was correctly assembled and represented as a single species.

Example 2 Characterization of BiXAb-6567 by Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) was used to compare the thermal stability of BiXAb-6567, stock anti-CD38 mAb, and stock anti-PD-L1 mAb. Differential scanning calorimetry experiments were performed using a Microcal™ VP-Capillary DSC system (Malvern Instruments).

All samples were centrifuged (20,000x g, 5 min, 4°C) and quantified for protein content prior to DSC analysis using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) using an IgG assay program. For the study, all samples were diluted in PBS to a final concentration of 1 mg/ml.

The time to equilibrium was 3 minutes, and thermograms were obtained in the range from 20 to 110°C at a scan rate of 60°C/hour, a filter period of 25 seconds and medium feedback (medium feedback). Prior to sample analysis, 5 buffer/buffer scans were measured to stabilize the instrument, with a buffer/buffer scan performed between each protein/buffer scan. The results were consistent with a non-2-state unfolding model with pre- and post-transition adjusted by subtracting the base.

The DSC curves shown in Fig. 3 (range 50 to 100°C) demonstrated the way in which individual Fv regions can lead to different Fab unfolding profiles; this experiment also demonstrated that the Fv regions are responsible for the observed stability of Fabs. The DSC profile of the anti-CD38 mAb showed two transitions: a large peak having a Cp max of 170 kcal/mol/°C and a Tm1 of 70.9°C corresponding to the unfolding of both CH2 and Fab domains, and a small peak having a Cp max of 20 kcal/ mol/°C and Tm2 81.5°C, corresponding to the unfolding of the CH3 domain. The DSC profile of the anti-PD-L1 mAb showed two transitions: a small peak having a Cp max of 20 kcal/mol/°C and a Tm1 of 69.9°C corresponding to the unfolding of the CH2 domain, and a large peak having a Cp max of 160 kcal/mol/°C. °C and Tm2 83.4°C corresponding to the unfolding of both CH3 and Fab domains.

The DSC profile of BiXAb-6567 also showed two transitions with two large peaks. The first peak had a Cp max of 130 kcal/mol/°C and a Tm1 of 71.5°C and corresponded to the unfolding of the CH2 and Fab domains of the anti-CD38 mAb; the second peak had a Cp max of 170 kcal/mol/°C and a Tm2 of 81.5°C and corresponded to the unfolding of the CH3 and Fab domains of the anti-PD-L1 mAb. Thus, the DSC profile of BiXAb-6567 resembled a superposition of two DSC profiles of two parent mAbs and illustrated the excellent assembly and stability of BiXAb-6567. The toneset of BiXAb-6567 (63.3°C) was similar to that of the parent mAbs (anti-CD38 Tonset=63.5°C and anti-PD-L1 Tonset=63.2°C), demonstrating that BiXAb-6567 had stability parameters similar to those of the original antibodies. The calculated ΔH of BiXAb-6567 was 1560 kcal/mol, reflecting the larger size of the bispecific molecule relative to the two parent antibodies (anti-CD38 ΔH=963 kcal/mol and anti-PD-L1 ΔH=820 kcal/mol).

Definitions:

Tm or denaturation/melting temperature is the point at which the concentration of unfolded and folded molecules is equal and is the midpoint of unfolding conformational transitions. As a parameter, it describes the sensitivity of a protein to thermal denaturation and thus is related to protein stability. The higher the Tm, the more stable the protein.

Tonset is the temperature at which the unfolding conformational transition begins. Values for this parameter are typically 5-10°C lower than Tm. It is also a parameter describing the stability of a protein, but also applies to resistance to thermal denaturation.

ΔH is the calorimetric enthalpy of unfolding, which reflects the destruction of intramolecular interactions in a protein (i.e., the destruction of intra- and inter-domain interactions). The thermal unfolding process is endothermic and therefore gives positive enthalpy values. The calorimetric enthalpy (ΔH) is the area under the peak of an unfolding thermal conformational transition.

Example 3 Cell Free Binding Properties of BiXAb-6567

Direct Plate ELISA CD38 Antigen Binding Assay

100 μl of either stock mAb, anti-CD38 or anti-PDL1, each at 3 μg/ml, prepared by dilution with PBS pH 7.4, was used to coat Maxisorp plates at 4°C overnight. Also, BiXAb-6567, at a concentration of 5 μg/ml, prepared by dilution with PBS pH 7.4, was used to coat Maxisorp plates at 4°C overnight. The plates were washed 5 times with 1x PBS containing 0.05% Tween-20 (PBST) and then blocked with 200 μl/well 1% BSA in 1x PBS at room temperature for 2 hours. The plates were then washed 5 times with 1x PBST. A seven-point 3-fold dilution series of recombinant His/Flag-tagged CD38 (Creative Biomart) in 1x PBS was prepared, starting at 1 μg/ml; 100 μl of each dilution step was added per well. The plates were incubated at room temperature for 1 hour 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 at room temperature for 1 hour. After 5 washes with 1x PBST, 100 μl/well of TMB substrate in 1x PBS was added to obtain colorimetric data and the plates were incubated for 15 minutes at room temperature for color to appear. The assay results were read using a Victor2 microplate reader (Perkin Elmer) at 650 nm.

BiXAb-6567 showed a dose-dependent binding curve very similar to that of the parent anti-CD38 antibody (FIG. 4A). The EC50 values of PD-L1 binding for both antibodies were as follows: EC50[BiXAb-6567] = 171 ng/mL and EC50[anti-CD38] = 199 ng/mL. This result suggests that BiXAb-6567 has properly assembled anti-CD38 Fab domains, as it showed binding similar to that of the original anti-CD38 mAb. The original anti-PDL1 mAb used as a negative control did not show any binding as expected.

Direct ELISA assay for PDL1 antigen binding.

100 μl of biotinylated human PD-L1 protein (AcroBiosystems) at 1 μg/ml prepared by dilution with 1x PBS pH7.4 was used to coat Maxisorp plates at 4°C overnight. The plates were washed 5 times with PBST, and then blocked with 200 μl/well of 1% BSA in 1x PBS at room temperature for 2 hours. The plates were then washed 5 times with 1x PBST. Seven-point 3-fold dilution series of either 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; 100 μl of each dilution step was added per well. The plates were incubated at room temperature for 1 hour 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 at room temperature for 1 hour. After 5 washes with 1x PBST, 100 μl/well of TMB substrate in 1x PBS was added to obtain colorimetric data and the plates were incubated for 15 minutes at room temperature for color to appear. The assay results were read using a microplate reader (Perkin Elmer) at 650 nm.

BiXAb-6567 showed a dose-dependent binding curve very similar to that of the parent anti-PD-L1 antibody (FIG. 4B). The EC50 values of PD-L1 binding for both antibodies were as follows: EC50[BiXAb-6567] = 93 ng/mL and EC50[anti-PD-L1] = 72 ng/mL. This result suggests that BiXAb-6567 has properly assembled anti-PD-L1 Fab domains as it exhibited similar binding to the parent anti-PD-L1 mAb. The original anti-CD38 mAb used as a negative control did not show any binding as expected.

ELISA two antigen binding assay

100 μl of recombinant human Fc-labeled CD38 (Creative BioMart) at 2 μg/ml prepared by dilution with 1x PBS pH7.4 was used to coat Maxisorp plates at 4°C overnight. The plates were washed 5 times with 1x PBST and then blocked with 200 μl/well of 1% BSA in 1x PBS at room temperature for 2 hours. The plates were washed 5 times with 1x PBST. Prepared a series of seven-point triplicate dilutions in 1x PBS BiXAb-6567 (starting at 1 μg/ml) and added 100 μl of each dilution step per well. The plates were incubated at room temperature for 1 hour, then washed 5 times with 1x PBST. Then 100 μl/well of 1 μg/ml biotinylated human PD-L1 (AcroBiosystems) in 1x PBS was added and the plates were incubated at room temperature for 1 hour. After 5 washes with 1x PBST, 100 μl/well of 0.1 μg/ml streptavidin-conjugated HRP (Biotechne) diluted with 1x PBS was added. The plates were incubated at room temperature for 1 hour. After 5 washes with 1x PBST, 100 μl/well of TMB substrate in 1x PBS was added to obtain colorimetric data and the plates were incubated for 15 minutes at room temperature for color to appear. The assay results were read using a Victor2 microplate reader (Perkin Elmer) at 650 nm.

BiXAb-6567 showed a dose dependent binding curve in a dual ELISA format suggesting that it has properly assembled anti-CD38 and anti-PD-L1 Fab domains (FIG. 4C). Thus, BiXAb-6567 was shown to be a bispecific antibody capable of simultaneously binding CD38 and PD-L1 with an EC50=144 ng/mL. The two parent mAbs, anti-CD38 or anti-PDL1, did not show any binding in this dual ELISA format as expected.

Example 4 Determination of Relative Binding Activity by Fluorescent Activated Cell Sorting (FACS)

CHO-CD38 cells (CHO cells stably transfected with full length human CD38) were cultured in DMEM-Glutamax-I 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 100 medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum.

3x10 ⁵ cells (CHO-CD38 or SKOV-3 or RPMI-8226) were used per sample. Cells were washed with 1x PBA solution (PBS supplemented with 1% BSA and 0.05% Na-azide). To determine FACS profiles, cells were stained with the corresponding antibodies at a concentration of 50 μg/ml in a volume of 30 μl. For titration of BiXAb-6567 and the original anti-CD38 antibody and subsequent determination of CHO-CD38 binding parameters, cells were stained with the corresponding antibodies at the indicated concentrations in a volume of 30 μl. Cells were incubated for 30 minutes on ice and then washed 2 times with 1 ml PBA solution. Cells were incubated with fluorescently labeled anti-human kappa or anti-human IgG Fc gamma-specific secondary antibodies on ice in the dark for 30 minutes and then washed 2 times with 1 ml PBA solution; at the end, the cells were resuspended in a final volume of 500 µl of PBA solution. Samples were analyzed using an Epics-XL or Navios flow cytometer (Beckman Coulter). In each experiment, 10,000 events were collected.

The binding profiles of BiXAb-6567 and the parent anti-CD38 and anti-PD-L1 parent antibodies are shown in FIG. 5A-C. We chose to test a multiple myeloma cell line, RPMI-8226, which express high levels of CD38 and low levels of PD-L1 (Fig. 5A); a CHO-CD38 cell line that expressed very high levels of CD38 due to stably transfected full-length CD38 (FIG. 5B); and the ovarian cancer cell line SKOV-3 known to express PD-L1 (FIG. 5C). These profiles showed one peak for BiXAb-6567, which was very similar to the profiles of both parental antibodies on 3 cell lines. This suggests that BiXAb-6567 is properly folded and has binding properties similar to those of the original antibodies. As expected, CHO-CD38 only expressed CD38 and did not express PD-L1, while SKOV-3 only expressed PD-L1 and did not express CD38.

In order to quantify that the binding properties of BiXAb-6567 are similar to those of the parent anti-CD38 antibody, titrations of BiXAb-6567 and anti-CD38 parent antibody were performed using CHO-CD38 cells as shown in FIG. 6. The EC50 of BiXAb-6567 was found to be 17.1 nM and that of the parent anti-CD38 was 8.5 nM, confirming the similar binding properties of the anti-CD38 Fab domains in BiXAb-6567 and the parent anti-CD38 antibody. The negative controls in this experiment, anti-PD-L1 and anti-CD20 antibodies, showed no binding to CHO-CD38 cells, as expected.

Example 5 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) with Unfractionated Non-Preactivated Mononuclear Cells (MNC)

CHO-CD38, SKOV-3 and RPMI-8226 cells were cultured as described in Example 4 above.

The following procedure was used to obtain MNC. Freshly drawn peripheral blood was treated with citrate to prevent clotting. Then 5 ml of Ficoll-Paque PLUS solution was layered with 6 ml of anticoagulant treated whole blood. Samples were centrifuged for 20 minutes at 2500 rpm at RT without subsequent disruption by centrifugation. MNC was collected from the plasma/ficoll interface. The suspension of MNC cells was diluted 1:10 in PBS and centrifuged for 5 minutes at 1800 rpm at room temperature. The supernatant was removed and the erythrocytes were lysed by adding 45 ml of ice-cold distilled water to the cell suspension for 30 seconds, after which 5 ml of 10x PBS was added. Cells were centrifuged for 5 minutes at 1800 rpm at room temperature and washed with 1x PBS three times to remove platelets. Finally, the cells were resuspended in 5 ml of cell culture medium. The number of cells was adjusted to achieve a ratio of 40:1 = effector cells: tumor cells in ADCC analysis.

For the ADCC test with the removal of radioactive ⁵¹ chromium 1x10 ⁶ target cells (RPMI 8226, SKOV-3 or CHO-CD38) were incubated with 100 microcuries ⁵¹ chromium in 200 μl PBS for 2 hours at 37°C and 5% CO2. After 2 hours of incubation, the cells were washed three times with 7 ml of medium and finally resuspended at a concentration of 0.1 x 10 ⁶ cells/ml. Target cells (5,000 cells/well) and MNCs in the presence of antibodies were incubated in a 96-well microtiter plate (sample volume 200 µl) for 3 hours at 37°C and 5% CO2. Triton X-100 was added to determine the maximum target cell lysis (=maximum counts per minute (cpm)) . To determine the base release of ⁵¹ chromium (= base cpm), the target cells were not subjected to further manipulations. After 4 hours of incubation, the microtiter plates were centrifuged for 5 minutes at 2000 rpm and 25 μl of the supernatant was mixed with 125 μl of Optiphase Supermix (Perkin Elmer) and incubated on an incubator shaker for 1 minute. Samples were analyzed using a MicroBeta TriLux beta particle counter (Perkin Elmer). Target cell lysis was calculated using the following formula:

% lysis = (experimental cpm - base cpm)/(max cpm - base cpm) x 100.

All measurements were carried out three times.

ADCC analysis of CD38+ cells (RPMI-8226 and CHO-CD38) was performed using non-preactivated MNC as effector cells (FIGS. 7 and 8). 0.8 nM and 0.3 nM, respectively; on CHO-CD38 cells, the cytotoxicity of BiXAb-6567 and anti-CD38 antibodies was EC50 0.2 nM and 0.07 nM, respectively. Anti-PD-L1 showed minimal activity on both cell lines; the two negative control mAbs, anti-CD20 and anti-HER2, did not promote lysis as expected. These results demonstrate the strong ADCC activity of BMX-6567 against CD38+ cells, which is similar to that of the original anti-CD38 antibody.

Example 6 ADCC Using Enriched Preactivated NK Cells

SKOV3, RPMI 8226, and CHO-CD38 cells were cultured as described in Example 4. MNCs were obtained as described in Example 5. NK cells were separated from MNCs by negative selection using the "Human NK Cell Isolation Kit" (Miltenyi) according to the manufacturer's instructions. NK cells were cultured overnight at a seeding density of 2x10 ⁶ cells/ml in RPMI supplemented with 10% fetal bovine serum. Added IL-12 or IL-15 to a final concentration of 10 ng/ml. ADCC assays were performed as already indicated in Example 5, except that the Effector Cell:Tumor Cell ratio was maintained at 10:1 and the reaction time was reduced to 3 hours.

The ADCC properties of the anti-PD-L1 fragment of BiXAb-6567 were analyzed on the PD-L1+ cell line SKOV-3 using enriched NK cells pre-activated with either IL-12 or IL-15. The results are shown in FIG. 9 and 10. This experiment compared the ADCC properties of BiXAb-6567 with those of the parent anti-PD-L1 antibody; an anti-HER2 antibody was used as a positive control, and an anti-CD20 antibody and a parent anti-CD38 antibody were used as negative controls, because SKOV-3 cells are PD-L1+/HER2+/CD20-/CD38-. Fig. 9 and 10 show the strong ADCC activity of BiXAb-6567 and the original anti-PD-L1 antibodies, regardless of whether IL-12 or IL-15 was used in NK cell culture. The EC50 values of BiXAb-6567 and the original anti-PD-L1 antibodies were 0.007 nM and 0.03 nM, respectively, when using IL-12. The profiles were even more similar when using IL-15; however, the plotted curves did not converge, thus preventing the calculation of EC50 values. These results demonstrate the strong ADCC activity of BMX-6567 against PD-L1+ cells, which is similar to that of the original anti-PD-L1 antibody.

--->

SEQUENCE LISTING

<110> BIOMUNEX Pharmaceuticals

<120> Stable bispecific antibodies

<130> B2466

<160> 21

<170>PatentIn version 3.5

<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 acids

<220>

<221> misc_feature

<222>(8)..(8)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (10)..(11)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (24)..(25)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (28)..(28)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (30)..(30)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (32)..(32)

<223> Xaa can be any naturally occurring amino acids

<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 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 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 (40)

1. A protein construct that provides for the formation of appropriate pairs of heavy/light chains and prevents their mismatch, which contains at least one mutated Fab fragment, which is selected from a) Fab fragment consisting of VH and VL domains of the antibody of interest; a CH1 domain derived from an immunoglobulin CH1 domain by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and a CL domain derived from an immunoglobulin CL domain by replacing the aspartic acid at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue; and b) Fab-fragment, consisting of VH and VL domains of the antibody of interest; a CH1 domain derived from an immunoglobulin CH1 domain by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue; and a CL domain derived from an immunoglobulin CL domain by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing the serine residue at position 176 of said CL domain with a valine residue. 2. The protein construct according to claim 1, which contains or consists of a multispecific antigen-binding fragment containing at least two Fab fragments with different CH1 and CL domains, each Fab fragment recognizing a different epitope of interest and these Fab fragments are located sequentially in any order, the C-terminal region of the CH1 domain of the first Fab fragment is connected to the N-terminal region of the VH domain of the next Fab fragment via a polypeptide linker, and at least one Fab fragment consists of the specified mutated Fab fragment, preferably two Fab fragments are mutated Fab fragments. 3. A protein construct according to claim 2, which is a multispecific antibody having two identical antigen-binding arms, each of which consists of the specified multispecific antigen-binding fragment. 4. The protein construct according to claim 3, which has an immunoglobulin-like structure containing two identical antigen-binding arms, each of which consists of the specified multispecific antigen-binding fragment; dimerized CH2 and CH3 immunoglobulin domains; an IgA, IgG, or IgD hinge region linking the C-terminal regions of the CH1 domains of the antigen-binding arms to the N-terminal regions of the CH2 domains. 5. Protein construct according to claim 4, which is preferably a bispecific antibody and contains at least two heavy chains and four light chains, with each heavy chain containing a) an immunoglobulin Fc region containing a hinge-CH2-CH3 domain, b) wherein the Fc region is linked to the CH1-VH heavy chain Fab of antibody 1 (Ab1) via said hinge domain, c) which in turn is connected to the CH1-VH heavy chain Fab of antibody 2 (Ab2) by a polypeptide linker sequence, and the polypeptide linker sequence connects the N-terminus of said Ab1 VH domain heavy chain Fab to the C-terminus of said Ab2 CH1 domain, and four light chains contain CL-VL Ab1 light chain Fabs and CL-VL Ab2 light chain Fabs linked to their heavy chain cognate domains; moreover, Ab1 and Ab2 recognize different epitopes, and wherein the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain derived from an immunoglobulin CH1 domain by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and the related CL domain is a mutated domain derived from an immunoglobulin CL domain by replacing a residue asparagine at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue, and/or wherein the Fab CH1 domain of one or the other of Ab1 or Ab2 is a mutated domain derived from an immunoglobulin CH1 domain by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue, and related CL the domain is a mutated domain derived from an immunoglobulin CL domain by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing the serine residue at position 176 of said CL domain with a valine residue. 6. The protein construct of claim 5, wherein the Fab CH1 domain of Ab1 is a mutated domain derived from an immunoglobulin CH1 domain by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and the related CL domain is a mutated domain derived from the CL domain of an immunoglobulin by replacing the asparagine residue at position 137 of said CL domain with a lysine residue and replacing 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 the immunoglobulin CH1 domain by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue, and the related CL domain is a mutated domain derived from an immunoglobulin CL domain by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing a serine residue at position 176 of the specified CL domain for the remainder to valine. 7. The protein construct according to claim 5, wherein the Fab CH1 domain of Ab2 is a mutated domain derived from an immunoglobulin CH1 domain by replacing the threonine residue at position 192 of said CH1 domain with aspartic acid, and the related CL domain is a mutated domain derived from the CL domain of an immunoglobulin by replacing the asparagine residue at position 137 of said CL domain with a lysine residue and replacing the serine residue at position 114 of said CL domain with an alanine residue, and wherein the Fab CH1 domain of Ab1 is a mutated domain derived from the immunoglobulin CH1 domain by replacing the leucine residue at position 124 of said CH1 domain with glutamine and replacing the serine residue at position 188 of said CH1 domain with a valine residue, and the related CL domain is a mutated domain derived from an immunoglobulin CL domain by replacing the valine residue at position 133 of said CL domain with a threonine residue and replacing a serine residue at position 176 of the specified CL domain for the remainder to valine. 8. Protein structure according to any one of paragraphs. 2-7, in which the sequence of the polypeptide linker contains or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO:1), where X ₁ , X ₂ , X _{3 ,} X ₄ , X _{5 ,} X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, are any amino acid. 9. The protein construct of claim 8, wherein the polypeptide linker sequence contains or consists of a sequence selected from the group consisting of EPKSCDKTHTSPPAPAPE LLGGPGGPPGPGPGGG (SEQ ID NO: 2); EPKSCDKTHTSPPAPAPE LLGGPAAPPAPAPAGG (SEQ ID NO: 3); EPKSCDKTHTSPPAPAPE LLGGPAAPPGPAPGGG (SEQ ID NO: 4); EPKSCDKTHTCPPCPAPE LLGGPSTPPTPSPSGG (SEQ ID NO:5) and EPKSCDKTHTSPPSPAPE LLGGPSTPPTPSPSGG (SEQ ID NO:6). 10. Protein structure according to any one of paragraphs. 1-9, recognizing both EGFR and HER2/neu. 11. A protein construct according to claim 10, which is a multispecific antibody, preferably a bispecific antibody, wherein Ab1 and Ab2, which are different, are independently selected from the group consisting of cetuximab or a mutated derivative thereof on the one hand, and trastuzumab or a mutated thereof derivative, on the other hand. 12. Protein structure according to any one of paragraphs. 1-9, recognizing both CD38 and PD-L1. 13. A protein construct according to claim 12, which is a multispecific antibody, preferably a bispecific antibody, wherein Ab1 and Ab2, which are different, are independently selected from the group consisting of daratumumab or a mutated derivative thereof on the one hand, and atezolizumab or a mutated derivative thereof derivative, on the other hand. 14. Protein construct according to claim 12, which is a bispecific antibody containing, preferably consisting of a) two heavy chains, each of which contains, preferably consists of SEQ ID NO: 7 and b) four light chains, two of which contain , preferably consist of SEQ ID NO: 15, and the other two contain, preferably consist of SEQ ID NO: 18. 15. The method of obtaining a protein construct according to paragraphs. 2-14, said method includes the following steps: a) culturing in a suitable medium and culturing conditions a host cell expressing the heavy chain of an antibody according to any one of paragraphs. 2-14 and the light chain of an antibody according to any one of paragraphs. 2-14; and b) recovering said obtained antibodies from the culture medium or from said cultured cells.

Images