HK40112971A - Egfr and lag3 dual targeted bispecific antibody and uses thereof - Google Patents

Abstract

Disclosed herein are bispecific antibodies against EGFR and LAG3, nucleic acids comprising the antibodies, vectors comprising the nucleic acids, and host cell comprising the nucleic acids or the vectors. Also disclosed are pharmaceutical compositions and antibody-drug conjugates comprising the antibodies, and therapeutic methods for using the antibodies.

Description

Bispecific antibodies targeting both EGFR and LAG3 and their applications

Invention Field

This application relates to bispecific antibodies targeting EGFR and LAG3, and the uses of such antibodies, particularly their use in cancer treatment.

Background Technology

Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. It consists of a kinase-active C-terminal intracellular region, an N-terminal extracellular receptor binding site, and a hydrophobic transmembrane domain. EGFR is a receptor for tyrosine kinases (RTKs). RTKs play a crucial role in controlling fundamental cellular processes such as cell circulation, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. EGFR can regulate a wide range of cellular processes via tyrosine kinase-regulated signal transduction pathways. Furthermore, EGFR activation leads to the simultaneous activation of multiple signal transduction cascades, including the MAPK pathway, the protein kinase C (PKC) pathway, and the PI(3)K-activated AKT pathway.

EGFR is frequently overexpressed or mutated in human tumors and contributes to tumor growth. Approximately 30% of solid tumors show gain-of-function genetic alterations in EGFR, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. Some cells exhibit "proto-oncogene addiction," a concept introduced by Weinstein in 2002, because they rely on the EGFR signaling pathway for survival and growth. Therefore, targeting EGFR could be a very effective treatment approach. Various strategies targeting EGFR and blocking the EGFR signaling pathway have been reported, including cetuximab and panitumumab. All of these strategies confirm that targeting EGFR is an effective anti-tumor therapy.

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered in an experiment designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines. It is a 70 kDa transmembrane protein structurally homologous to CD4 with four extracellular glycosylation sites. LAG3 is not expressed in resting peripheral blood lymphocytes but is expressed in activated T cells, NK cells, some B cells, and dendritic cells. Like CD4, LAG3 binds to MHC class II molecules with high affinity. However, unlike CD4, LAG3 does not interact with the human immunodeficiency virus gp120 protein. Studies using soluble LAG3 immunoglobulin fusion protein (sLAG3Ig) have shown that LAG3 binds directly and specifically to MHC class II molecules on the cell surface.

The cytoplasmic tail of LAG3 consists of three conserved motifs, one of which is a highly unique and conserved six-amino acid sequence (KIEELE, SEQ ID NO:20) that has been shown to be essential for LAG3-mediated T cell function downregulation. The interaction of LAG3 with its ligands (MHC class II molecules expressed on antigen-presenting cells, such as macrophages and dendritic cells) inhibits the activation of T cells and NK cells, suppressing their ability to recognize and kill cancer cells. Furthermore, CD4 ⁺ CD25 ⁺ regulatory T cells (T _reg ) have been shown to express LAG3 upon activation, and antibodies against LAG3 have inhibited the inhibition induced by induced T _reg cells both in vitro and in vivo, suggesting that LAG3 contributes to the repressor activity of T _reg cells.

The roles of EGFR and LAG3 in cancer are well-established, and while monotherapy targeting either EGFR or LAG3 may initially be highly effective in cancer treatment, resistance often develops. Therefore, there is a need for bispecific antibodies that target both EGFR and LAG3, which are predicted to achieve effective cancer treatment by recognizing both antigens simultaneously.

Invention Overview

This disclosure provides recombinant EGFR×LAG3 bispecific antibodies designed to bind simultaneously to surface antigens on target cells and certain immune cells, such as T cells. Numerous functional assays have demonstrated the potent antitumor effects of antibodies engineered in the form of EGFR×LAG3 bispecific antibodies against a variety of cancers, particularly EGFR-positive cancers such as colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer.

In one aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises the amino acid sequence shown in SEQ ID NO:4; VH1 comprises the amino acid sequence shown in SEQ ID NO:8; VL2 comprises the amino acid sequence shown in SEQ ID NO:12; and VH2 comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from the N-terminus to the C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from the N-terminus to the C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some embodiments, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2 and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2 and IgG4 (e.g., human IgG1, IgG2 and IgG4).

In some implementations, CL is derived from the λ light chain or the κ light chain.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5, and preferably the linker comprises an amino acid sequence as shown in SEQ ID NO:19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:18.

In another aspect, this disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

In another aspect, this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

In another respect, this disclosure provides pharmaceutical compositions comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides conjugates comprising the bispecific antibody or its antigen-binding fragment disclosed herein, and the chemical portion conjugated thereto.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In another aspect, this disclosure provides a method of treating a subject with cancer, including administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject's cancer.

In another aspect, this disclosure provides for the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

In some embodiments of the uses disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Attached Figure Description

The features and advantages of the invention can be understood by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments utilizing the principles of the invention. In the drawings:

Figure 1 schematically shows the structure of the EGFR×LAG3 bispecific antibody (BsAb) CMD005.

Figure 2A shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human EGFR as measured by ELISA.

Figure 2B shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human LAG3 as measured by ELISA.

Figure 3A shows the binding of EGFR×LAG3 BsAb CMD005 to the EGFR-expressing cell line Caco-2, as measured by flow cytometry.

Figure 3B shows the binding of EGFR×LAG3 BsAb CMD005 to LAG3 on the surface of activated T cells, as measured by flow cytometry.

Figure 4A shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between EGFR and EGF.

Figure 4B shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between LAG3 and FGL1.

Figure 5 shows the ability of EGFR×LAG3 BsAb CMD005, as measured by flow cytometry, to block the interaction between LAG3 and MHC class II molecules expressed in Raji cells.

Figure 6A shows the growth inhibition of A431 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6B shows the growth inhibition of Caco-2 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6C shows the growth inhibition of Achn cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 7 shows the plasma concentrations of EGFR×LAG3 BsAb CMD005 in mice treated with CMD005 at 15, 39, 63, and 87 hours post-treatment.

Figure 8A shows the inhibition of EGFR×LAG3BsAb CMD005 on MC38-hEGFR tumor growth in hLAG3-c57 BL/6 mice by tumor volume measurement.

Figure 8B shows the body weight of hLAG3-c57 BL/6 mice in all groups during the tumor growth inhibition assay.

Invention Details

The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the following detailed description of the embodiments.

The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended to provide a general understanding of the invention. These embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are denoted using similar reference numerals throughout the specification.

Unless otherwise stated or defined, all terms used have the common meaning in the art that is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. (eds.), “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; and Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), vols. 1-3, Cold Spring Harbor Laboratory Press. (1989); F. Ausubel et al., eds., “Current protocols in molecular biology”, Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology” (6th edition), Mosby/Elsevier, Edinburgh (2001); and Janeway et al., “Immunology” (6th edition), Garland Science Publishing/Churchill Livingstone, New York (2005), and the general background techniques cited above.

definition

As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

Unless otherwise stated or defined, the term “comprising” and its variations such as “including” and “containing” should be understood to mean that the said element or step or group of elements or steps is included, but does not exclude any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

The “light chain variable region (VL)” or “heavy chain variable region (VH)” consists of a “framework” region interspersed with three “complementarity-determining regions” or “CDRs”. The framework region is used to modulate the CDRs to specifically bind to antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.

The amino acid sequence of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia Kabat CDR, where CDR-H1 is a complex of Chothia CDR and Kabat CDR; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat.

Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five types (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain.

As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense and includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of one or more antibodies that retains the ability to specifically bind antigens. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody.

Examples of antigen-binding fragments covered by the term "antigen-binding moiety" of antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; (vii) dAb fragments, which consist of a VH domain; (viii) separate complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked using recombination methods through synthetic linkers that enable them to be made into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies should also be covered under the term "antigen-binding fragment" in the term antibody. Additionally, the term also includes "linear antibodies," which comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) forming the antigen-binding region together with a complementary light chain polypeptide, and any modified versions of the aforementioned fragments that retain antigen-binding activity.

These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. Affinity, expressed as the equilibrium constant (KD) for antibody-antigen dissociation, is a measure of the strength of binding between the antigenic determinant and the antibody's antigen-binding site: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1/KD.

Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity involves the affinity between an antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies bind to antigens with a dissociation constant (KD) of ^10⁻⁵ to ^10⁻¹² M or less, preferably ^10⁻⁷ to ^10⁻¹² M or less, more preferably ^10⁻⁸ to ^10⁻¹² M, and/or with a binding affinity of at least ^{10⁷ M⁻¹} , preferably at least ^{10⁸ M⁻¹} , more preferably at least ^{10⁹ M⁻¹} , such as at least ^{10¹² M⁻¹} . Any _KD value greater than ^10⁻⁴ M is generally considered to indicate nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatcherd analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations known per se in the art.

The term "epitope" refers to the site on an antigen where an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically consist of at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antibodies and are therefore specific targets for antibodies or their antigen-binding fragments.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percentage of identity between the amino acid sequence and SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gappedBLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid linked to it.

As used in this article, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutical acceptable” means that the carrier or excipient is compatible with the other components of the composition and does not cause significant toxicity to the recipient, and/or that such carrier or excipient is approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms “treatment,” “management,” etc., refer to the administration of a drug or procedure to achieve an effect. These effects may be preventative in terms of the complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of the partial or complete cure of a disease and/or its symptoms. As used herein, “treatment” may include the treatment of a disease or condition (e.g., cancer) in mammals, particularly humans, and includes: (a) the prevention of the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases that may be related to or caused by the primary disease); (b) the suppression of the disease, i.e., the halting of its development; and (c) the alleviation of the disease, i.e., the remission of the disease. Treatment may refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter such as elimination; alleviation; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of deterioration or decline; or making the endpoint of deterioration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of disease, disease symptoms or disease side effects in a subject.

As used in this article, the term "effective amount" refers to an amount that is sufficient to treat a disease when administered to a subject to treat that disease.

As used herein, the term “subject” means any mammalian subject for whom a diagnosis, treatment, or therapy is intended. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

Bispecific antibodies

This disclosure provides a bispecific antibody and its antigen-binding fragment, comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some implementations, the CDR sequence is defined according to the Kabat numbering system.

When the CDR sequence is defined according to the Kabat numbering system, the VL1 of the antibody disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 1 (QASQDISNYLN), SEQ ID NO: 2 (DASNLET), and SEQ ID NO: 3 (QHFDHLPLA), respectively, and the VH1 of the antibody disclosed herein comprises HCDR1, HCDR2, and H having the amino acid sequences shown in SEQ ID NO: 5 (SGDYYWT), SEQ ID NO: 6 (HIYYSGNTNYNPSLKS), and SEQ ID NO: 7 (DRVTGAFDI), respectively. CDR3; The VL2 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 9 (SGSSSNIGSNAVS), SEQ ID NO: 10 (YDDLLPS) and SEQ ID NO: 11 (AAWDDSLNAFV), respectively, and the VH2 of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 13 (SYGIS), SEQ ID NO: 14 (WISAYNGNTNYAQKLQG) and SEQ ID NO: 15 (DSSSWWVDAFDI), respectively.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 4, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VH1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 8, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VL2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 12, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3. In some embodiments, VH2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 16, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In the context of functional variants, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

In some implementations, insertions, deletions, and/or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

In a preferred embodiment, VL1 contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:8; VL2 contains the amino acid sequence shown in SEQ ID NO:12; and VH2 contains the amino acid sequence shown in SEQ ID NO:16.

The bispecific antibodies disclosed herein may contain an Fc region, which includes the antibody's CH2 and CH3 regions.

The Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one implementation, the Fc region is defective in effector function. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector functions such as ADCC. Such mutations have been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001).

In one embodiment, the Fc region contains a mutation that removes the receptor site for Asn-linked glycosylation or is otherwise manipulated to alter the glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in one specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.

In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC can be optimized using the method described by et al. (1999) Nature Biotech 17:176. In a further embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100:2411.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from N-terminus to C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some implementations, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2, and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4). In a preferred embodiment, each of CH1, CH2, and CH3 is independently derived from IgG1.

In some embodiments, CH1-CH2-CH3 comprises an amino acid sequence as shown in any one of SEQ ID NO:21-23. In a preferred embodiment, CH1-CH2-CH3 comprises an amino acid sequence as shown in SEQ ID NO:21.

In some implementations, CL is derived from the λ light chain or the κ light chain. In a preferred implementation, CL is derived from the κ light chain.

In some embodiments, CL comprises an amino acid sequence as shown in any one of SEQ ID NO:24-25. In a preferred embodiment, CL comprises an amino acid sequence as shown in SEQ ID NO:24.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5. In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGS (SEQ ID NO: 26). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGS (SEQ ID NO: 27). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 19). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 29). In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 17 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR and/or LAG3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 18 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In some embodiments, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, even more preferably 5% to 30%, even more preferably 10% to 25%, even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the insertion, deletion, and/or substitution can be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH3.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitution have been described above.

In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18.

Nucleic acid

This disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

The term "nucleic acid" encompasses single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or any modified form of nucleotides. These modifications include base modifications, such as uridine bromide and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and modifications involving nucleotide linkages, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

In some embodiments, the nucleic acid is a ribonucleotide (RNA) or a deoxyribonucleotide (DNA). In some embodiments, the present invention provides a ribonucleotide (RNA) comprising a nucleotide sequence encoding a bispecific antibody disclosed herein. In some embodiments, the present invention provides a deoxyribonucleotide comprising a deoxynucleotide sequence encoding a bispecific antibody disclosed herein.

In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

In some embodiments, the ribonucleotide (RNA) may be introduced into human cells in vivo. In some embodiments, the ribonucleotide (RNA) of the present invention is contained in a carrier or delivery agent.

carrier

This disclosure provides vectors containing the nucleic acids disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy or light chain variable region of a bispecific antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

Any vector may be suitable for use in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs containing a replication system functional in prokaryotic or eukaryotic host cells can be prepared. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

For example, the vector could be an adenovirus vector containing a nucleotide sequence encoding the bispecific antibody disclosed herein. This vector could be administered to a subject and subsequently enter cells within the subject, thereby integrating the nucleotide sequence encoding the bispecific antibody disclosed herein into the cell's genome, and subsequently, the cell would express the bispecific antibody disclosed herein.

host cells

This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium. The bacteria include *Serratia* (e.g., *Serratia marcescans*), *Shigella*, *Bacilli* (e.g., *B. subtilis* and *B. licheniformis*), *Pseudomonas* (e.g., *P. aeruginosa*), and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or in vivo. The host cells of the present invention can be administered to a subject in vivo, and the host cells express the bispecific antibodies disclosed herein in vivo.

The present invention provides host cells in which any of the above-described vectors have been introduced. The invention further provides a method for preparing the bispecific antibody of the present invention, wherein the method comprises a) culturing the host cells of the fourth aspect of the present invention under conditions suitable for the production of bispecific antibodies; and b) obtaining the bispecific antibody from the culture.

Pharmaceutical Composition

This disclosure provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and pharmaceutically acceptable carriers or excipients.

Bispecific antibodies or their antigen-binding fragments, or the pharmaceutical agents of the present invention (also referred to herein as "active ingredients") and their derivatives, fragments, analogs, and homologs, may be incorporated into pharmaceutical compositions for ease of administration. Such compositions typically comprise a bispecific antibody or its antigen-binding fragment or a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with the use of the drug. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and anhydrous media (such as fixed oils) may also be used. The use of such media and pharmaceutical agents for the active pharmaceutical ingredient is well known in the art. The use of any conventional media or pharmaceutical agent in the composition is considered except where it is incompatible with the active compound. Supplemental active compounds may also be incorporated into the composition.

In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate; and reagents for adjusting pH, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (wherein being water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL ^™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the desired amount of the active compound, along with one or a combination of the ingredients listed above, into a suitable solvent as needed, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of preparing sterile powders for sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce a powder containing the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binder and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavorings.

For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a sprayer.

Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for mucosal application. Mucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion known in the art.

The active compound can also be formulated as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

This invention provides therapeutic compositions comprising the bispecific antibody of the invention or an antigen-binding fragment thereof. The therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in formulations known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN ^™ ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm SciTechnol 52:238-311.

Conjugate

This disclosure provides conjugates comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the chemical moiety conjugated thereto.

In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as “immunoconjugates.” When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an “antibody-drug conjugate” or “ADC.”

The term "conjugation" or "linkage" refers to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

The chemical moiety can be linked to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent linkages can be used. The procedure for linking the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to generate binding to the chemical moiety. Alternatively, the antibody may be derivatized to expose or link additional reactive functional groups. Derivatization may involve linking any of a number of known adapter molecules. The adapter can be any molecule used to link the antibody to the chemical moiety. The adapter is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable adapters are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon adapters, heterocyclic carbon adapters, or peptide adapters. In the case where the antibody and chemical moiety are peptides, the adapter can be linked via their side groups to the constituent amino acids (e.g., via a disulfide bond with cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

In some cases, when an immunoconjugate reaches its target site, it is desirable to release the chemical portion from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.

Conditions experienced by enzymatically active or immunoconjugates within or near target cells may induce linker cleavage to release the chemical portion from the antibody.

Given the numerous methods already reported for linking various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art will be able to determine suitable methods for linking a given reagent to an antibody or other peptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally linked (through chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a dimeric antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (such as the streptavidin core region or a multihistidine tag).

One type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types). Suitable cross-linking agents include heterobifunctional cross-linking agents having two distinctly reactive groups separated by a suitable spacer (such as m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional cross-linking agents (such as disuccinimide octanoate). Such linkers are commercially available.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be, for example, maytansine, galdromycin, a microtubule inhibitor such as a microtubule binding agent (e.g., olistatin), or a minor groove binding agent such as chaziomycin.

Other suitable therapeutic agents include small molecule cytotoxic agents, which are compounds with a molecular weight of less than 700 Daltons capable of killing mammalian cells. These compounds may also contain toxic metals that produce cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, which are compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, chalcogenide, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porphyrin sodium photosensitizer II, temozolomide, topotecan, trimesartan gluconate, olprestatin E, vincristine, and doxorubicin; and peptide cytotoxic agents, which are proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, Dnase, and RNase; radionuclides, which are unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying. Examples include iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents can be used to promote the binding of these radionuclides to molecules or polymers thereof.

In some embodiments, the detectable portion may be selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Detectable portions for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

Bispecific antibodies can be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance transduction (MTR), ultrasound, fiber optic examination, and laparoscopy. Specifically, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used.

Bispecific antibodies or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a bispecific antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding an additional reagent that typically produces a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Bispecific antibodies or antigen-binding fragments can also be conjugated to biotin and detected indirectly by binding to avidin or streptavidin. It should be noted that avidin itself can bind to enzymes or fluorescent labels.

Bispecific antibodies can be fused with self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned to the ends of constant regions. HaloTag is a self-labeled protein tag derived from bacterial enzymes (haloalkane dehalogenases) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligands contain chloroalkane linkers attached to fluorophores such as near-infrared fluorophores (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

Bispecific antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanide elements (such as europium and dysprosium) and manganese.

Paramagnetic particles, such as superparamagnetic iron oxide, can also be used as labels. Bispecific antibodies can also be labeled with predetermined peptide epitopes recognized by a second reporter molecule (such as leucine zipper pairs, binding sites of the second antibody, metal-binding domains, or epitope tags). In some embodiments, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

Bispecific antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, and 131I.

In some implementations, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine, such as IL-2 and IFN-γ; a chemokine, such as IL-8; platelet-4; melanoma growth-stimulating protein; a complement activator; a viral/bacterial protein domain; or a viral/bacterial peptide.

Treatment

This disclosure provides a method for treating a subject with cancer, comprising administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer.

Examples of cancer include: leukemia, such as, but not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and myelodysplastic syndromes); chronic leukemia, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; polycythemia vera; lymphoma, such as, but not limited to, Hodgkin's disease and non-Hodgkin's disease; and multiple myeloma, such as, but not limited to... Smoking multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; unexplained monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, and soft tissue sarcoma. Angiosarcoma, (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, glioblastoma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pinealoblastoma, primary brain lymphoma; breast cancer, This includes, but is not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid carcinoma, medullary thyroid adenoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma; GIST-gastrointestinal stromal tumors; and pancreatic cancer, such as, but not limited to, islet tumors, gastrinomas, glucagonomas, vasodilator intestinal peptide tumors, and somatostatin-secreting tumors. Cancers include: ovarian tumors and carcinoid tumors or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas like iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancers. Cancers, such as but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancers, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancers, such as but not limited to adenocarcinoma, mycosis fungoides, ulcerative, superficially spreading, widely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancers, such as but not limited to hepatocellular carcinoma and hepatoblastic carcinoma; gallbladder cancers, such as but not limited to adenocarcinoma; bile duct cancers, such as but not limited to breast cancers. Head-shaped, nodular, and diffuse cholangiocarcinoma; lung cancer, such as non-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer (SCLC); testicular cancer, such as, but not limited to, germ cell tumors, seminoma, anaplastic, classic (typical), spermatogenic cell tumors, non-seminomatous tumors, embryonal carcinoma, teratoma, and choriocarcinoma (yolk sac tumor); prostate cancer, such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers, such as penile cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as, but not limited to, Limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and warts; skin cancer, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigines melanoma, and acral lentigines melanoma; renal cancer, such as, but not limited to, renal cell carcinoma, clear cell renal cell carcinoma, adenocarcinoma, adrenoidoma, fibrosarcoma, and transitional cell carcinoma (renal pelvis and/or ureter); Wilms' tumor; bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioma endothelial sarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. Preferably, the cancer is selected from breast cancer, melanoma, prostate cancer, ovarian cancer, colorectal cancer, lung cancer, or glioma.

In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some implementations, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment can depend on many factors, such as antibody activity and the route of administration.

A single dose of the antibody, composition, or conjugate described herein may be administered to mammals once or in a series of sub-dose over appropriate time periods, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the antibody, composition, or conjugate may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more sub-dose administrations per day.

The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, antibodies, compositions, or conjugates are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the antibody, composition, or conjugate disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

Medical Use

This disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject’s cancer.

This disclosure also provides the disclosed bispecific antibodies or antigen-binding fragments thereof, the disclosed pharmaceutical compositions, or the disclosed conjugates for use in treating cancer in subjects.

In some embodiments of the uses disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Reagent test kit

This disclosure provides pharmaceutical kits or reagent kits comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as the bispecific antibodies or antigen-binding fragments disclosed herein. Optionally, associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for manufacture, use, or sale for human administration.

In one embodiment, the kit includes a first container of the bispecific antibody disclosed herein. In another embodiment, the kit includes a first container which is a vial containing the bispecific antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

In one embodiment, this document provides an injection device comprising a bispecific antibody. In one embodiment, the injection device comprises a bispecific antibody in a sterile solution. In another embodiment, the injection device is a syringe.

Example

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, are representative of preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Variations and other uses covered by the spirit of the invention as defined by the claims will be apparent to those skilled in the art.

MC38-hEGFR cancer cells were purchased from Kyinno Biotechnology Co., Ltd. Activated T cells were obtained by activating frozen PBMCs with human CD3/CD28 T cell activators. Other tumor cell lines, including human epidermal cancer cell line A431, human lymphoma cell line Raji, human colon cancer cell line Caco-2, and renal cancer cell line Achn, were purchased from ATCC.

Human EGF protein and human FGL1 protein were purchased from ACROBiosystems. Human EGFR/HER1/ErbB1 protein and human LAG3 protein were purchased from Sino Biological. Anti-human IgG (γ chain specific)-R-PE antibody, anti-human IgG (Fc specific)-peroxidase antibody, and monoclonal anti-peroxidase were purchased from Sigma. PE anti-His tag antibody was purchased from BioLegend. Anti-EGFR mAb and anti-LAG3 mAb were prepared in-house.

The full light chain and heavy chain sequences of the Anti-EGFR mAb and Anti-LAG3 mAb are shown below.

Anti-EGFR mAb

Heavy chain:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34) light chain:

DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:35)

Anti-LAG3 mAb

Heavy chain:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDSSSWWVDAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:36)

Light chain:

SYELTQPPSVSEAPRQRVTISSCSGSSSNIGSNAVSWYQQVPRKAPKLLVYYDDLLPSGVSDRFSGSKSGTSASLAIRGLQSEDEADYYCAAWDDSLNAFVFGTGTKVTVLGQPK ANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:37)

Example 1. Construction and preliminary characterization of EGFR×LAG3 bispecific antibody

Cloning of EGFR×LAG3 bispecific antibody (BsAb)

The light chain of the EGFR×LAG3 BsAb consists of an anti-LAG-3VL and a CL domain. The heavy chain, from the N-terminus to the C-terminus, contains an anti-LAG-3VH, a CH1 domain, a wild-type human IgG1 Fc, and an anti-EGFR single-chain variable region fragment (ScFv). The structure of the EGFR×LAG3 BsAb (also referred to herein as CMD005) is shown in Figure 1. The light chain of both the anti-EGFR ScFv and the EGFR×LAG3 BsAb was synthesized directly by GENEWIZ. The following primers, synthesized by GENEWIZ, were used to generate the EGFR×LAG3 BsAb construct:

Vector-HC-FP:5’TAATTCTAGAGTCGACAATCAACCTCTGG 3’(Justice) (SEQ ID NO:30);

Vector-HC-RP:5’TGATCCACCACCTCCACTACCG 3’(antense) (SEQ ID NO:31);

EGFR-HB-pa-VH-FP:

5’GTGGTGGATCACAGGTGCAGCTGCAGGAGAG 3’(Justice) (SEQ ID NO:32);

EGFR-HB-pa-VL-RP:

5’GATTGTCGACTCTAGAATTATTTGATTTCCACCTTGGTTCCG3’ (antisense) (SEQ ID NO:33).

To generate EGFR×LAG3 BsAb, anti-EGFR ScFv was fused to the C-terminus of the LAG-3 monoclonal antibody via a (G4S)4 linker. The light and heavy chains were separately constructed into a single vector pcDNA3.4 for mammalian cell expression.

Protein expression, purification and preliminary characterization

EGFR×LAG3 BsAb was expressed in CHO-S cells using the ExpiFectamine CHO transfection kit. Cells continued to grow for 5–7 days post-transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The supernatant containing the target protein was loaded onto a gravity column equipped with EshmunoA (Merck). Subsequent purification was performed according to the manufacturer's instructions.

EGFR×LAG3 BsAb was well expressed in transiently transfected CHO-S cells and secreted into the culture supernatant. On non-reducing SDS-PAGE, the apparent molecular weight (aMW) of CMD005 was approximately 76.3 kDa. On reduced SDS-PAGE, the heavy and light chains were close together, with an apparent molecular weight of approximately 23.1 kDa (data not shown).

The CDR sequence of CMD005 according to the Kabat numbering system is shown in Table 1. The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of CMD005 are shown in Table 2. The complete light chain and heavy chain sequences of CMD005 are shown in Table 3.

Table 1. CDR Sequence of CMD005

Table 2. VL and VH sequences of CMD005

Table 3. Heavy and light chain sequences of CMD005

Example 2. Binding of EGFR×LAG3 BsAb CMD005 with recombinant EGFR and LAG3

ELISA was performed according to standard protocol to determine the binding affinity of CMD005 for recombinant EGFR and LAG3. Briefly, human antigens EGFR or LAG3 were coated at 100 ng per well overnight at 4°C on Corning EIA/RIA high-binding 96-well plates (Corning Inc.), and blocked with PBS (pH 7.4, MPBS) containing 3% skim milk, 3% bovine serum albumin, and 1‰ Tween 20, respectively. Antibodies were added in 5-fold serial dilutions and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected using HRP-conjugated streptavidin (Sino Biological). This assay was developed using TMB substrates (Solarbio) at room temperature and monitored using a microplate reader at 450 nm. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1 kappa (IgG1κ) isotype control was used as a negative control. The results are shown in Figures 2A-2B.

The results showed that CMD005 could bind to recombinant human EGFR with an EC50 of 0.43 nM and recombinant human LAG3 with an EC50 of 0.09 nM. Furthermore, there was no significant difference in binding activity between the EGFR×LAG3 bispecific antibody CMD005 and anti-EGFR or anti-LAG3 mAbs.

Example 3: EGFR×LAG3 BsAb CMD005 binding to cell surface-associated EGFR and LAG3

To measure the binding ability of EGFR×LAG3 BsAb CMD005 to LAG3-associated cells on the cell surface, flow cytometry was performed using activated T cells, and the binding ability of CMD005 to EGFR-associated cells on the cell surface was measured by flow cytometry using the human colon cancer cell line Caco-2. Approximately 5 × ^10⁵ cells were incubated on ice for 1 hour with five-fold serially diluted antibody. Cells were washed once with PBS containing 0.5% bovine serum albumin (PBSA) and resuspended in 100 μL of PBSA. Then, 1 μL of anti-human IgG (γ-chain specific)-R-phycoerythrin antibody ((Sigma)) generated in goats was added and incubated for 30 minutes. Cells were washed once with PBSA and then used for flow cytometry analysis. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 3A-3B.

The results showed that EGFR×LAG3 BsAb CMD005 could bind well to Caco-2 and T cells, and the binding affinity was similar to that of anti-EGFR mAb or anti-LAG3 mAb.

Example 4. Blocking of EGFR-EGF, LAG3-FGL1, and LAG3-MHCII binding mediated by EGFR×LAG3 BsAb CMD005

ELISA assays were performed to determine the ability of EGFR×LAG3 BsAb CMD005 to block the binding of EGFR/EGF and LAG3/FGL1 receptor-ligand pairs.

Human antigen EGFR was coated at 500 ng per well on Corning EIA/RIA high-binding 96-well plates (Corning Inc.) at 4 °C overnight and blocked with 3% MPBS (pH 7.4). Five serially diluted antibodies were mixed with human antigen EGF (14 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected by HRP anti-6X antibody (Abcam). This assay was developed using TMB substrate (Solarbio) at room temperature and monitored at 450 nm using a microplate reader. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1κ isotype control was used as a negative control. Results are shown in Figure 4A.

Human antigen FGL1 was coated at 100 ng per well on plates overnight at 4°C and blocked with PBS containing 3% bovine serum albumin and 1‰ Tween 20. Five serially diluted antibodies were mixed with human antigen LAG3 (4 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The remaining steps were the same as above. Human IgG1κ isotype control was used as a negative control. The results are shown in Figure 4B.

The blocking ability of CMD005 against the LAG3/MHCII receptor-ligand pair was measured using cell-based competitive flow cytometry. Approximately 5 × ^10⁵ Raji cells expressing MHCII on their cell surface were incubated on ice for 1 h with serially diluted antibody solution and biotin-labeled human antigen LAG3 (400 ng). Cells were washed once with 0.5% PBSA and then resuspended in 100 μL of PBSA. Then, 0.1% streptavidin R-PE conjugate (Invitrogen) was added and the cells were incubated on ice for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. A human IgG1κ isotype control was used as a negative control. The results are shown in Figure 5.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong blocking effect on the receptor-ligand interaction of EGFR/EGF and LAG3/FGL1, with EC50 values of 1.86 nM and 32.85 nM, respectively (Figures 4A-4B). Furthermore, CMD005 inhibited or competed with MHCII for binding to LAG3, with an EC50 value of 6.88 nM (Figure 5).

Example 5. EGFR×LAG3 BsAb CMD005-mediated killing of human cancer cell lines

The in vitro efficacy of EGFR×LAG3 BsAb CMD005 was evaluated using several cancer cell lines, including A431, Caco-2, and Achn, via CCK8 assay. These three cell lines were centrifuged and resuspended in RPMI 1640 complete medium, then added to 96-well round-bottom plates at densities of 1000, 4000, and 1500 cells/100 μL, respectively. Cells were incubated at 37°C with 5% _CO2 supplementation for 24 hours. The next day, 100 μL of antibody solution (serially diluted 10-fold from 100 nM) was added to each well accordingly. After 4–10 days of incubation (cell confluence 80–90%), the medium was removed from the target cells, and 100 μL of RPMI 1640 complete medium containing 10% CCK-8 was added, followed by incubation in a _CO2 incubator for 30 minutes. Cell inhibitory activity was measured using a microplate reader according to the manufacturer's instructions. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 6A-6C.

The results showed that EGFR×LAG3 BsAb CMD005 exhibited potent inhibition of tumor cell growth in a dose-dependent manner. There was no significant difference in the inhibitory effect of EGFR×LAG3 BsAb CMD005 on tumor cell growth compared to anti-EGFR mAb.

Example 6. Inhibition of tumor growth in mice mediated by anti-AXL bispecific antibody

Measurement of pharmacokinetics in mice

On day 0, two BALB/c mice were intravenously administered 200 μg of EGFR×LAG3 BsAb CMD005. Plasma samples were collected at 15, 39, 63, and 87 hours post-treatment for measuring serum antibody concentrations by ELISA, and a standard curve was generated using the original antibody stock. The results are shown in Figure 7.

The results showed that the serum concentration of CMD005 gradually decreased, but remained at a high level at the endpoint.

In vivo tumor growth inhibition

The in vivo efficacy of EGFR×LAG3 BsAb CMD005 was evaluated in hLAG3-c57 BL/6 mice. Three × ^10⁶ MC38-hEGFR tumor cells were subcutaneously seeded into the right abdomen of hLAG3-c57 BL/6 mice. After identifying palpable tumors, mice were randomized according to tumor volume and body weight. A negative control group was administered 10 mg/kg of human IgG1κ isotype intravenously. Experimental groups included an anti-EGFR mAb group (10 mg/kg), an anti-LAG3 mAb group (10 mg/kg), an anti-EGFR mAb + anti-LAG3 mAb group (10 + 10 mg/kg), and a CMD005 group (10 mg/kg). These mice were administered the drugs twice weekly. After three weeks of treatment, the mice were sacrificed and tumor volume was measured. The tumor growth inhibition (TGI) rate was calculated using the following formula: TGI(%) = [1 - ( _TT0 )/( _CC0 )] × 100 (T and _T0 : tumor volumes on the specific experimental day and the randomized experimental day, respectively; C and _C0 : the corresponding average tumor volumes in the control group). A value > 100% indicates tumor shrinkage. The results are shown in Figures 8A-8B.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong inhibitory effect on tumor growth, stronger than that of anti-EGFR mAb, anti-LAG3 mAb, and anti-EGFR mAb + anti-LAG3 mAb (Figure 8A). The tumor growth inhibition rate was 137.1% in the CMD005 group (10 mg/kg), 27.1% in the anti-EGFR mAb group (10 mg/kg), 86.2% in the anti-LAG3 mAb group (10 mg/kg), and 60.2% in the anti-EGFR mAb + LAG3 mAb group (10 + 10 mg/kg). There were only minor changes in body weight in all groups of mice (Figure 8B).

In summary, in vivo studies have shown that EGFR×LAG3 BsAb CMD005 has a long serum half-life and can specifically and effectively inhibit tumor growth. Therefore, this bispecific antibody therapy, EGFR×LAG3 BsAb CMD005, has great potential for clinical development.

While preferred embodiments of the invention have been shown and described herein, such embodiments will be apparent to those skilled in the art only as examples. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The appended claims are intended to define the scope of the invention and to cover the methods and structures within the scope of these claims and their equivalents.

Invention Field

This application relates to bispecific antibodies targeting EGFR and LAG3, and the uses of such antibodies, particularly their use in cancer treatment.

Background Technology

Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. It consists of a kinase-active C-terminal intracellular region, an N-terminal extracellular receptor binding site, and a hydrophobic transmembrane domain. EGFR is a receptor for tyrosine kinases (RTKs). RTKs play a crucial role in controlling fundamental cellular processes such as cell circulation, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. EGFR can regulate a wide range of cellular processes via tyrosine kinase-regulated signal transduction pathways. Furthermore, EGFR activation leads to the simultaneous activation of multiple signal transduction cascades, including the MAPK pathway, the protein kinase C (PKC) pathway, and the PI(3)K-activated AKT pathway.

EGFR is frequently overexpressed or mutated in human tumors and contributes to tumor growth. Approximately 30% of solid tumors show gain-of-function genetic alterations in EGFR, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. Some cells exhibit "proto-oncogene addiction," a concept introduced by Weinstein in 2002, because they rely on the EGFR signaling pathway for survival and growth. Therefore, targeting EGFR could be a very effective treatment approach. Various strategies targeting EGFR and blocking the EGFR signaling pathway have been reported, including cetuximab and panitumumab. All of these strategies confirm that targeting EGFR is an effective anti-tumor therapy.

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered in an experiment designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines. It is a 70 kDa transmembrane protein structurally homologous to CD4 with four extracellular glycosylation sites. LAG3 is not expressed in resting peripheral blood lymphocytes but is expressed in activated T cells, NK cells, some B cells, and dendritic cells. Like CD4, LAG3 binds to MHC class II molecules with high affinity. However, unlike CD4, LAG3 does not interact with the human immunodeficiency virus gp120 protein. Studies using soluble LAG3 immunoglobulin fusion protein (sLAG3Ig) have shown that LAG3 binds directly and specifically to MHC class II molecules on the cell surface.

The cytoplasmic tail of LAG3 consists of three conserved motifs, one of which is a highly unique and conserved six-amino acid sequence (KIEELE, SEQ ID NO:20) that has been shown to be essential for LAG3-mediated T cell function downregulation. The interaction of LAG3 with its ligands (MHC class II molecules expressed on antigen-presenting cells, such as macrophages and dendritic cells) inhibits the activation of T cells and NK cells, suppressing their ability to recognize and kill cancer cells. Furthermore, CD4 ⁺ CD25 ⁺ regulatory T cells (T _reg ) have been shown to express LAG3 upon activation, and antibodies against LAG3 have inhibited the inhibition induced by induced T _reg cells both in vitro and in vivo, suggesting that LAG3 contributes to the repressor activity of T _reg cells.

The roles of EGFR and LAG3 in cancer are well-established, and while monotherapy targeting either EGFR or LAG3 may initially be highly effective in cancer treatment, resistance often develops. Therefore, there is a need for bispecific antibodies that target both EGFR and LAG3, which are predicted to achieve effective cancer treatment by recognizing both antigens simultaneously.

Invention Overview

This disclosure provides recombinant EGFR×LAG3 bispecific antibodies designed to bind simultaneously to surface antigens on target cells and certain immune cells, such as T cells. Numerous functional assays have demonstrated the potent antitumor effects of antibodies engineered in the form of EGFR×LAG3 bispecific antibodies against a variety of cancers, particularly EGFR-positive cancers such as colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer.

In one aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises the amino acid sequence shown in SEQ ID NO:4; VH1 comprises the amino acid sequence shown in SEQ ID NO:8; VL2 comprises the amino acid sequence shown in SEQ ID NO:12; and VH2 comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from the N-terminus to the C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from the N-terminus to the C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some embodiments, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2 and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2 and IgG4 (e.g., human IgG1, IgG2 and IgG4).

In some implementations, CL is derived from the λ light chain or the κ light chain.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5, and preferably the linker comprises an amino acid sequence as shown in SEQ ID NO:19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:18.

In another aspect, this disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

In another aspect, this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

In another respect, this disclosure provides pharmaceutical compositions comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides conjugates comprising the bispecific antibody or its antigen-binding fragment disclosed herein, and the chemical portion conjugated thereto.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In another aspect, this disclosure provides a method of treating a subject with cancer, including administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject's cancer.

In another aspect, this disclosure provides for the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

In some embodiments of the uses disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Attached Figure Description

The features and advantages of the invention can be understood by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments utilizing the principles of the invention. In the drawings:

Figure 1 schematically shows the structure of the EGFR×LAG3 bispecific antibody (BsAb) CMD005.

Figure 2A shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human EGFR as measured by ELISA.

Figure 2B shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human LAG3 as measured by ELISA.

Figure 3A shows the binding of EGFR×LAG3 BsAb CMD005 to the EGFR-expressing cell line Caco-2, as measured by flow cytometry.

Figure 3B shows the binding of EGFR×LAG3 BsAb CMD005 to LAG3 on the surface of activated T cells, as measured by flow cytometry.

Figure 4A shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between EGFR and EGF.

Figure 4B shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between LAG3 and FGL1.

Figure 5 shows the ability of EGFR×LAG3 BsAb CMD005, as measured by flow cytometry, to block the interaction between LAG3 and MHC class II molecules expressed in Raji cells.

Figure 6A shows the growth inhibition of A431 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6B shows the growth inhibition of Caco-2 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6C shows the growth inhibition of Achn cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 7 shows the plasma concentrations of EGFR×LAG3 BsAb CMD005 in mice treated with CMD005 at 15, 39, 63, and 87 hours post-treatment.

Figure 8A shows the inhibition of EGFR×LAG3BsAb CMD005 on MC38-hEGFR tumor growth in hLAG3-c57 BL/6 mice by tumor volume measurement.

Figure 8B shows the body weight of hLAG3-c57 BL/6 mice in all groups during the tumor growth inhibition assay.

Invention Details

The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the following detailed description of the embodiments.

The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended to provide a general understanding of the invention. These embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are denoted using similar reference numerals throughout the specification.

Unless otherwise stated or defined, all terms used have the common meaning in the art that is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. (eds.), “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; and Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), vols. 1-3, Cold Spring Harbor Laboratory Press. (1989); F. Ausubel et al., eds., “Current protocols in molecular biology”, Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology” (6th edition), Mosby/Elsevier, Edinburgh (2001); and Janeway et al., “Immunology” (6th edition), Garland Science Publishing/Churchill Livingstone, New York (2005), and the general background techniques cited above.

definition

As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

Unless otherwise stated or defined, the term “comprising” and its variations such as “including” and “containing” should be understood to mean that the said element or step or group of elements or steps is included, but does not exclude any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

The “light chain variable region (VL)” or “heavy chain variable region (VH)” consists of a “framework” region interspersed with three “complementarity-determining regions” or “CDRs”. The framework region is used to modulate the CDRs to specifically bind to antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.

The amino acid sequence of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia Kabat CDR, where CDR-H1 is a complex of Chothia CDR and Kabat CDR; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat.

Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five types (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain.

As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense and includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of one or more antibodies that retains the ability to specifically bind antigens. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody.

Examples of antigen-binding fragments covered by the term "antigen-binding moiety" of antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; (vii) dAb fragments, which consist of a VH domain; (viii) separate complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked using recombination methods through synthetic linkers that enable them to be made into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies should also be covered under the term "antigen-binding fragment" in the term antibody. Additionally, the term also includes "linear antibodies," which comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) forming the antigen-binding region together with a complementary light chain polypeptide, and any modified versions of the aforementioned fragments that retain antigen-binding activity.

These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. Affinity, expressed as the equilibrium constant (KD) for antibody-antigen dissociation, is a measure of the strength of binding between the antigenic determinant and the antibody's antigen-binding site: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1/KD.

Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity involves the affinity between an antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies bind to antigens with a dissociation constant (KD) of ^10⁻⁵ to ^10⁻¹² M or less, preferably ^10⁻⁷ to ^10⁻¹² M or less, more preferably ^10⁻⁸ to ^10⁻¹² M, and/or with a binding affinity of at least ^{10⁷ M⁻¹} , preferably at least ^{10⁸ M⁻¹} , more preferably at least ^{10⁹ M⁻¹} , such as at least ^{10¹² M⁻¹} . Any _KD value greater than ^10⁻⁴ M is generally considered to indicate nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatcherd analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations known per se in the art.

The term "epitope" refers to the site on an antigen where an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically consist of at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antibodies and are therefore specific targets for antibodies or their antigen-binding fragments.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percentage of identity between the amino acid sequence and SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gappedBLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid linked to it.

As used in this article, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutical acceptable” means that the carrier or excipient is compatible with the other components of the composition and does not cause significant toxicity to the recipient, and/or that such carrier or excipient is approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms “treatment,” “management,” etc., refer to the administration of a drug or procedure to achieve an effect. These effects may be preventative in terms of the complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of the partial or complete cure of a disease and/or its symptoms. As used herein, “treatment” may include the treatment of a disease or condition (e.g., cancer) in mammals, particularly humans, and includes: (a) the prevention of the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases that may be related to or caused by the primary disease); (b) the suppression of the disease, i.e., the halting of its development; and (c) the alleviation of the disease, i.e., the remission of the disease. Treatment may refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter such as elimination; alleviation; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of deterioration or decline; or making the endpoint of deterioration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of disease, disease symptoms or disease side effects in a subject.

As used in this article, the term "effective amount" refers to an amount that is sufficient to treat a disease when administered to a subject to treat that disease.

As used herein, the term “subject” means any mammalian subject for whom a diagnosis, treatment, or therapy is intended. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

Bispecific antibodies

This disclosure provides a bispecific antibody and its antigen-binding fragment, comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some implementations, the CDR sequence is defined according to the Kabat numbering system.

When the CDR sequence is defined according to the Kabat numbering system, the VL1 of the antibody disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 1 (QASQDISNYLN), SEQ ID NO: 2 (DASNLET), and SEQ ID NO: 3 (QHFDHLPLA), respectively, and the VH1 of the antibody disclosed herein comprises HCDR1, HCDR2, and H having the amino acid sequences shown in SEQ ID NO: 5 (SGDYYWT), SEQ ID NO: 6 (HIYYSGNTNYNPSLKS), and SEQ ID NO: 7 (DRVTGAFDI), respectively. CDR3; The VL2 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 9 (SGSSSNIGSNAVS), SEQ ID NO: 10 (YDDLLPS) and SEQ ID NO: 11 (AAWDDSLNAFV), respectively, and the VH2 of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 13 (SYGIS), SEQ ID NO: 14 (WISAYNGNTNYAQKLQG) and SEQ ID NO: 15 (DSSSWWVDAFDI), respectively.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 4, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VH1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 8, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VL2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 12, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3. In some embodiments, VH2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 16, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In the context of functional variants, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

In some implementations, insertions, deletions, and/or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

In a preferred embodiment, VL1 contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:8; VL2 contains the amino acid sequence shown in SEQ ID NO:12; and VH2 contains the amino acid sequence shown in SEQ ID NO:16.

The bispecific antibodies disclosed herein may contain an Fc region, which includes the antibody's CH2 and CH3 regions.

The Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one implementation, the Fc region is defective in effector function. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector functions such as ADCC. Such mutations have been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001).

In one embodiment, the Fc region contains a mutation that removes the receptor site for Asn-linked glycosylation or is otherwise manipulated to alter the glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in one specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.

In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC can be optimized using the method described by et al. (1999) Nature Biotech 17:176. In a further embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100:2411.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from N-terminus to C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some implementations, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2, and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4). In a preferred embodiment, each of CH1, CH2, and CH3 is independently derived from IgG1.

In some embodiments, CH1-CH2-CH3 comprises an amino acid sequence as shown in any one of SEQ ID NO:21-23. In a preferred embodiment, CH1-CH2-CH3 comprises an amino acid sequence as shown in SEQ ID NO:21.

In some implementations, CL is derived from the λ light chain or the κ light chain. In a preferred implementation, CL is derived from the κ light chain.

In some embodiments, CL comprises an amino acid sequence as shown in any one of SEQ ID NO:24-25. In a preferred embodiment, CL comprises an amino acid sequence as shown in SEQ ID NO:24.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5. In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGS (SEQ ID NO: 26). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGS (SEQ ID NO: 27). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 19). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 29). In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 17 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR and/or LAG3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 18 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In some embodiments, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, even more preferably 5% to 30%, even more preferably 10% to 25%, even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the insertion, deletion, and/or substitution can be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH3.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitution have been described above.

In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18.

Nucleic acid

This disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

The term "nucleic acid" encompasses single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or any modified form of nucleotides. These modifications include base modifications, such as uridine bromide and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and modifications involving nucleotide linkages, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

In some embodiments, the nucleic acid is a ribonucleotide (RNA) or a deoxyribonucleotide (DNA). In some embodiments, the present invention provides a ribonucleotide (RNA) comprising a nucleotide sequence encoding a bispecific antibody disclosed herein. In some embodiments, the present invention provides a deoxyribonucleotide comprising a deoxynucleotide sequence encoding a bispecific antibody disclosed herein.

In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

In some embodiments, the ribonucleotide (RNA) may be introduced into human cells in vivo. In some embodiments, the ribonucleotide (RNA) of the present invention is contained in a carrier or delivery agent.

carrier

This disclosure provides vectors containing the nucleic acids disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy or light chain variable region of a bispecific antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

Any vector may be suitable for use in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs containing a replication system functional in prokaryotic or eukaryotic host cells can be prepared. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

For example, the vector could be an adenovirus vector containing a nucleotide sequence encoding the bispecific antibody disclosed herein. This vector could be administered to a subject and subsequently enter cells within the subject, thereby integrating the nucleotide sequence encoding the bispecific antibody disclosed herein into the cell's genome, and subsequently, the cell would express the bispecific antibody disclosed herein.

host cells

This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium. The bacteria include *Serratia* (e.g., *Serratia marcescans*), *Shigella*, *Bacilli* (e.g., *B. subtilis* and *B. licheniformis*), *Pseudomonas* (e.g., *P. aeruginosa*), and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or in vivo. The host cells of the present invention can be administered to a subject in vivo, and the host cells express the bispecific antibodies disclosed herein in vivo.

The present invention provides host cells in which any of the above-described vectors have been introduced. The invention further provides a method for preparing the bispecific antibody of the present invention, wherein the method comprises a) culturing the host cells of the fourth aspect of the present invention under conditions suitable for the production of bispecific antibodies; and b) obtaining the bispecific antibody from the culture.

Pharmaceutical Composition

This disclosure provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and pharmaceutically acceptable carriers or excipients.

Bispecific antibodies or their antigen-binding fragments, or the pharmaceutical agents of the present invention (also referred to herein as "active ingredients") and their derivatives, fragments, analogs, and homologs, may be incorporated into pharmaceutical compositions for ease of administration. Such compositions typically comprise a bispecific antibody or its antigen-binding fragment or a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with the use of the drug. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and anhydrous media (such as fixed oils) may also be used. The use of such media and pharmaceutical agents for the active pharmaceutical ingredient is well known in the art. The use of any conventional media or pharmaceutical agent in the composition is considered except where it is incompatible with the active compound. Supplemental active compounds may also be incorporated into the composition.

In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate; and reagents for adjusting pH, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (wherein being water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL ^™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the desired amount of the active compound, along with one or a combination of the ingredients listed above, into a suitable solvent as needed, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of preparing sterile powders for sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce a powder containing the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binder and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavorings.

For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a sprayer.

Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for mucosal application. Mucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion known in the art.

The active compound can also be formulated as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

This invention provides therapeutic compositions comprising the bispecific antibody of the invention or an antigen-binding fragment thereof. The therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in formulations known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN ^™ ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm SciTechnol 52:238-311.

Conjugate

This disclosure provides conjugates comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the chemical moiety conjugated thereto.

In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as “immunoconjugates.” When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an “antibody-drug conjugate” or “ADC.”

The term "conjugation" or "linkage" refers to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

The chemical moiety can be linked to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent linkages can be used. The procedure for linking the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to generate binding to the chemical moiety. Alternatively, the antibody may be derivatized to expose or link additional reactive functional groups. Derivatization may involve linking any of a number of known adapter molecules. The adapter can be any molecule used to link the antibody to the chemical moiety. The adapter is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable adapters are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon adapters, heterocyclic carbon adapters, or peptide adapters. In the case where the antibody and chemical moiety are peptides, the adapter can be linked via their side groups to the constituent amino acids (e.g., via a disulfide bond with cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

In some cases, when an immunoconjugate reaches its target site, it is desirable to release the chemical portion from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.

Conditions experienced by enzymatically active or immunoconjugates within or near target cells may induce linker cleavage to release the chemical portion from the antibody.

Given the numerous methods already reported for linking various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art will be able to determine suitable methods for linking a given reagent to an antibody or other peptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally linked (through chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a dimeric antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (such as the streptavidin core region or a multihistidine tag).

One type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types). Suitable cross-linking agents include heterobifunctional cross-linking agents having two distinctly reactive groups separated by a suitable spacer (such as m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional cross-linking agents (such as disuccinimide octanoate). Such linkers are commercially available.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be, for example, maytansine, galdromycin, a microtubule inhibitor such as a microtubule binding agent (e.g., olistatin), or a minor groove binding agent such as chaziomycin.

Other suitable therapeutic agents include small molecule cytotoxic agents, which are compounds with a molecular weight of less than 700 Daltons capable of killing mammalian cells. These compounds may also contain toxic metals that produce cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, which are compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, chalcogenide, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porphyrin sodium photosensitizer II, temozolomide, topotecan, trimesartan gluconate, olprestatin E, vincristine, and doxorubicin; and peptide cytotoxic agents, which are proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, Dnase, and RNase; radionuclides, which are unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying. Examples include iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents can be used to promote the binding of these radionuclides to molecules or polymers thereof.

In some embodiments, the detectable portion may be selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Detectable portions for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

Bispecific antibodies can be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance transduction (MTR), ultrasound, fiber optic examination, and laparoscopy. Specifically, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used.

Bispecific antibodies or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a bispecific antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding an additional reagent that typically produces a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Bispecific antibodies or antigen-binding fragments can also be conjugated to biotin and detected indirectly by binding to avidin or streptavidin. It should be noted that avidin itself can bind to enzymes or fluorescent labels.

Bispecific antibodies can be fused with self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned to the ends of constant regions. HaloTag is a self-labeled protein tag derived from bacterial enzymes (haloalkane dehalogenases) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligands contain chloroalkane linkers attached to fluorophores such as near-infrared fluorophores (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

Bispecific antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanide elements (such as europium and dysprosium) and manganese.

Paramagnetic particles, such as superparamagnetic iron oxide, can also be used as labels. Bispecific antibodies can also be labeled with predetermined peptide epitopes recognized by a second reporter molecule (such as leucine zipper pairs, binding sites of the second antibody, metal-binding domains, or epitope tags). In some embodiments, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

Bispecific antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, and 131I.

In some implementations, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine, such as IL-2 and IFN-γ; a chemokine, such as IL-8; platelet-4; melanoma growth-stimulating protein; a complement activator; a viral/bacterial protein domain; or a viral/bacterial peptide.

Treatment

This disclosure provides a method for treating a subject with cancer, comprising administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer.

Examples of cancer include: leukemia, such as, but not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and myelodysplastic syndromes); chronic leukemia, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; polycythemia vera; lymphoma, such as, but not limited to, Hodgkin's disease and non-Hodgkin's disease; and multiple myeloma, such as, but not limited to... Smoking multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; unexplained monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, and soft tissue sarcoma. Angiosarcoma, (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, glioblastoma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pinealoblastoma, primary brain lymphoma; breast cancer, This includes, but is not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid carcinoma, medullary thyroid adenoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma; GIST-gastrointestinal stromal tumors; and pancreatic cancer, such as, but not limited to, islet tumors, gastrinomas, glucagonomas, vasodilator intestinal peptide tumors, and somatostatin-secreting tumors. Cancers include: ovarian tumors and carcinoid tumors or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas like iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancers. Cancers, such as but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancers, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancers, such as but not limited to adenocarcinoma, mycosis fungoides, ulcerative, superficially spreading, widely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancers, such as but not limited to hepatocellular carcinoma and hepatoblastic carcinoma; gallbladder cancers, such as but not limited to adenocarcinoma; bile duct cancers, such as but not limited to breast cancers. Head-shaped, nodular, and diffuse cholangiocarcinoma; lung cancer, such as non-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer (SCLC); testicular cancer, such as, but not limited to, germ cell tumors, seminoma, anaplastic, classic (typical), spermatogenic cell tumors, non-seminomatous tumors, embryonal carcinoma, teratoma, and choriocarcinoma (yolk sac tumor); prostate cancer, such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers, such as penile cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as, but not limited to, Limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and warts; skin cancer, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigines melanoma, and acral lentigines melanoma; renal cancer, such as, but not limited to, renal cell carcinoma, clear cell renal cell carcinoma, adenocarcinoma, adrenoidoma, fibrosarcoma, and transitional cell carcinoma (renal pelvis and/or ureter); Wilms' tumor; bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioma endothelial sarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. Preferably, the cancer is selected from breast cancer, melanoma, prostate cancer, ovarian cancer, colorectal cancer, lung cancer, or glioma.

In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some implementations, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment can depend on many factors, such as antibody activity and the route of administration.

A single dose of the antibody, composition, or conjugate described herein may be administered to mammals once or in a series of sub-dose over appropriate time periods, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the antibody, composition, or conjugate may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more sub-dose administrations per day.

The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, antibodies, compositions, or conjugates are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the antibody, composition, or conjugate disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

Medical Use

This disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject’s cancer.

This disclosure also provides the disclosed bispecific antibodies or antigen-binding fragments thereof, the disclosed pharmaceutical compositions, or the disclosed conjugates for use in treating cancer in subjects.

In some embodiments of the uses disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Reagent test kit

This disclosure provides pharmaceutical kits or reagent kits comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as the bispecific antibodies or antigen-binding fragments disclosed herein. Optionally, associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for manufacture, use, or sale for human administration.

In one embodiment, the kit includes a first container of the bispecific antibody disclosed herein. In another embodiment, the kit includes a first container which is a vial containing the bispecific antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

In one embodiment, this document provides an injection device comprising a bispecific antibody. In one embodiment, the injection device comprises a bispecific antibody in a sterile solution. In another embodiment, the injection device is a syringe.

Example

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, are representative of preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Variations and other uses covered by the spirit of the invention as defined by the claims will be apparent to those skilled in the art.

MC38-hEGFR cancer cells were purchased from Kyinno Biotechnology Co., Ltd. Activated T cells were obtained by activating frozen PBMCs with human CD3/CD28 T cell activators. Other tumor cell lines, including human epidermal cancer cell line A431, human lymphoma cell line Raji, human colon cancer cell line Caco-2, and renal cancer cell line Achn, were purchased from ATCC.

Human EGF protein and human FGL1 protein were purchased from ACROBiosystems. Human EGFR/HER1/ErbB1 protein and human LAG3 protein were purchased from Sino Biological. Anti-human IgG (γ chain specific)-R-PE antibody, anti-human IgG (Fc specific)-peroxidase antibody, and monoclonal anti-peroxidase were purchased from Sigma. PE anti-His tag antibody was purchased from BioLegend. Anti-EGFR mAb and anti-LAG3 mAb were prepared in-house.

The full light chain and heavy chain sequences of the Anti-EGFR mAb and Anti-LAG3 mAb are shown below.

Anti-EGFR mAb

Heavy chain:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34) light chain:

DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:35)

Anti-LAG3 mAb

Heavy chain:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDSSSWWVDAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:36)

Light chain:

SYELTQPPSVSEAPRQRVTISSCSGSSSNIGSNAVSWYQQVPRKAPKLLVYYDDLLPSGVSDRFSGSKSGTSASLAIRGLQSEDEADYYCAAWDDSLNAFVFGTGTKVTVLGQPK ANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:37)

Example 1. Construction and preliminary characterization of EGFR×LAG3 bispecific antibody

Cloning of EGFR×LAG3 bispecific antibody (BsAb)

The light chain of the EGFR×LAG3 BsAb consists of an anti-LAG-3VL and a CL domain. The heavy chain, from the N-terminus to the C-terminus, contains an anti-LAG-3VH, a CH1 domain, a wild-type human IgG1 Fc, and an anti-EGFR single-chain variable region fragment (ScFv). The structure of the EGFR×LAG3 BsAb (also referred to herein as CMD005) is shown in Figure 1. The light chain of both the anti-EGFR ScFv and the EGFR×LAG3 BsAb was synthesized directly by GENEWIZ. The following primers, synthesized by GENEWIZ, were used to generate the EGFR×LAG3 BsAb construct:

Vector-HC-FP:5’TAATTCTAGAGTCGACAATCAACCTCTGG 3’(Justice) (SEQ ID NO:30);

Vector-HC-RP:5’TGATCCACCACCTCCACTACCG 3’(antense) (SEQ ID NO:31);

EGFR-HB-pa-VH-FP:

5’GTGGTGGATCACAGGTGCAGCTGCAGGAGAG 3’(Justice) (SEQ ID NO:32);

EGFR-HB-pa-VL-RP:

5’GATTGTCGACTCTAGAATTATTTGATTTCCACCTTGGTTCCG3’ (antisense) (SEQ ID NO:33).

To generate EGFR×LAG3 BsAb, anti-EGFR ScFv was fused to the C-terminus of the LAG-3 monoclonal antibody via a (G4S)4 linker. The light and heavy chains were separately constructed into a single vector pcDNA3.4 for mammalian cell expression.

Protein expression, purification and preliminary characterization

EGFR×LAG3 BsAb was expressed in CHO-S cells using the ExpiFectamine CHO transfection kit. Cells continued to grow for 5–7 days post-transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The supernatant containing the target protein was loaded onto a gravity column equipped with EshmunoA (Merck). Subsequent purification was performed according to the manufacturer's instructions.

EGFR×LAG3 BsAb was well expressed in transiently transfected CHO-S cells and secreted into the culture supernatant. On non-reducing SDS-PAGE, the apparent molecular weight (aMW) of CMD005 was approximately 76.3 kDa. On reduced SDS-PAGE, the heavy and light chains were close together, with an apparent molecular weight of approximately 23.1 kDa (data not shown).

The CDR sequence of CMD005 according to the Kabat numbering system is shown in Table 1. The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of CMD005 are shown in Table 2. The complete light chain and heavy chain sequences of CMD005 are shown in Table 3.

Table 1. CDR Sequence of CMD005

Table 2. VL and VH sequences of CMD005

Table 3. Heavy and light chain sequences of CMD005

Example 2. Binding of EGFR×LAG3 BsAb CMD005 with recombinant EGFR and LAG3

ELISA was performed according to standard protocol to determine the binding affinity of CMD005 for recombinant EGFR and LAG3. Briefly, human antigens EGFR or LAG3 were coated at 100 ng per well overnight at 4°C on Corning EIA/RIA high-binding 96-well plates (Corning Inc.), and blocked with PBS (pH 7.4, MPBS) containing 3% skim milk, 3% bovine serum albumin, and 1‰ Tween 20, respectively. Antibodies were added in 5-fold serial dilutions and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected using HRP-conjugated streptavidin (Sino Biological). This assay was developed using TMB substrates (Solarbio) at room temperature and monitored using a microplate reader at 450 nm. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1 kappa (IgG1κ) isotype control was used as a negative control. The results are shown in Figures 2A-2B.

The results showed that CMD005 could bind to recombinant human EGFR with an EC50 of 0.43 nM and recombinant human LAG3 with an EC50 of 0.09 nM. Furthermore, there was no significant difference in binding activity between the EGFR×LAG3 bispecific antibody CMD005 and anti-EGFR or anti-LAG3 mAbs.

Example 3: EGFR×LAG3 BsAb CMD005 binding to cell surface-associated EGFR and LAG3

To measure the binding ability of EGFR×LAG3 BsAb CMD005 to LAG3-associated cells on the cell surface, flow cytometry was performed using activated T cells, and the binding ability of CMD005 to EGFR-associated cells on the cell surface was measured by flow cytometry using the human colon cancer cell line Caco-2. Approximately 5 × ^10⁵ cells were incubated on ice for 1 hour with five-fold serially diluted antibody. Cells were washed once with PBS containing 0.5% bovine serum albumin (PBSA) and resuspended in 100 μL of PBSA. Then, 1 μL of anti-human IgG (γ-chain specific)-R-phycoerythrin antibody ((Sigma)) generated in goats was added and incubated for 30 minutes. Cells were washed once with PBSA and then used for flow cytometry analysis. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 3A-3B.

The results showed that EGFR×LAG3 BsAb CMD005 could bind well to Caco-2 and T cells, and the binding affinity was similar to that of anti-EGFR mAb or anti-LAG3 mAb.

Example 4. Blocking of EGFR-EGF, LAG3-FGL1, and LAG3-MHCII binding mediated by EGFR×LAG3 BsAb CMD005

ELISA assays were performed to determine the ability of EGFR×LAG3 BsAb CMD005 to block the binding of EGFR/EGF and LAG3/FGL1 receptor-ligand pairs.

Human antigen EGFR was coated at 500 ng per well on Corning EIA/RIA high-binding 96-well plates (Corning Inc.) at 4 °C overnight and blocked with 3% MPBS (pH 7.4). Five serially diluted antibodies were mixed with human antigen EGF (14 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected by HRP anti-6X antibody (Abcam). This assay was developed using TMB substrate (Solarbio) at room temperature and monitored at 450 nm using a microplate reader. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1κ isotype control was used as a negative control. Results are shown in Figure 4A.

Human antigen FGL1 was coated at 100 ng per well on plates overnight at 4°C and blocked with PBS containing 3% bovine serum albumin and 1‰ Tween 20. Five serially diluted antibodies were mixed with human antigen LAG3 (4 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The remaining steps were the same as above. Human IgG1κ isotype control was used as a negative control. The results are shown in Figure 4B.

The blocking ability of CMD005 against the LAG3/MHCII receptor-ligand pair was measured using cell-based competitive flow cytometry. Approximately 5 × ^10⁵ Raji cells expressing MHCII on their cell surface were incubated on ice for 1 h with serially diluted antibody solution and biotin-labeled human antigen LAG3 (400 ng). Cells were washed once with 0.5% PBSA and then resuspended in 100 μL of PBSA. Then, 0.1% streptavidin R-PE conjugate (Invitrogen) was added and the cells were incubated on ice for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. A human IgG1κ isotype control was used as a negative control. The results are shown in Figure 5.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong blocking effect on the receptor-ligand interaction of EGFR/EGF and LAG3/FGL1, with EC50 values of 1.86 nM and 32.85 nM, respectively (Figures 4A-4B). Furthermore, CMD005 inhibited or competed with MHCII for binding to LAG3, with an EC50 value of 6.88 nM (Figure 5).

Example 5. EGFR×LAG3 BsAb CMD005-mediated killing of human cancer cell lines

The in vitro efficacy of EGFR×LAG3 BsAb CMD005 was evaluated using several cancer cell lines, including A431, Caco-2, and Achn, via CCK8 assay. These three cell lines were centrifuged and resuspended in RPMI 1640 complete medium, then added to 96-well round-bottom plates at densities of 1000, 4000, and 1500 cells/100 μL, respectively. Cells were incubated at 37°C with 5% _CO2 supplementation for 24 hours. The next day, 100 μL of antibody solution (serially diluted 10-fold from 100 nM) was added to each well accordingly. After 4–10 days of incubation (cell confluence 80–90%), the medium was removed from the target cells, and 100 μL of RPMI 1640 complete medium containing 10% CCK-8 was added, followed by incubation in a _CO2 incubator for 30 minutes. Cell inhibitory activity was measured using a microplate reader according to the manufacturer's instructions. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 6A-6C.

The results showed that EGFR×LAG3 BsAb CMD005 exhibited potent inhibition of tumor cell growth in a dose-dependent manner. There was no significant difference in the inhibitory effect of EGFR×LAG3 BsAb CMD005 on tumor cell growth compared to anti-EGFR mAb.

Example 6. Inhibition of tumor growth in mice mediated by anti-AXL bispecific antibody

Measurement of pharmacokinetics in mice

On day 0, two BALB/c mice were intravenously administered 200 μg of EGFR×LAG3 BsAb CMD005. Plasma samples were collected at 15, 39, 63, and 87 hours post-treatment for measuring serum antibody concentrations by ELISA, and a standard curve was generated using the original antibody stock. The results are shown in Figure 7.

The results showed that the serum concentration of CMD005 gradually decreased, but remained at a high level at the endpoint.

In vivo tumor growth inhibition

The in vivo efficacy of EGFR×LAG3 BsAb CMD005 was evaluated in hLAG3-c57 BL/6 mice. Three × ^10⁶ MC38-hEGFR tumor cells were subcutaneously seeded into the right abdomen of hLAG3-c57 BL/6 mice. After identifying palpable tumors, mice were randomized according to tumor volume and body weight. A negative control group was administered 10 mg/kg of human IgG1κ isotype intravenously. Experimental groups included an anti-EGFR mAb group (10 mg/kg), an anti-LAG3 mAb group (10 mg/kg), an anti-EGFR mAb + anti-LAG3 mAb group (10 + 10 mg/kg), and a CMD005 group (10 mg/kg). These mice were administered the drugs twice weekly. After three weeks of treatment, the mice were sacrificed and tumor volume was measured. The tumor growth inhibition (TGI) rate was calculated using the following formula: TGI(%) = [1 - ( _TT0 )/( _CC0 )] × 100 (T and _T0 : tumor volumes on the specific experimental day and the randomized experimental day, respectively; C and _C0 : the corresponding average tumor volumes in the control group). A value > 100% indicates tumor shrinkage. The results are shown in Figures 8A-8B.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong inhibitory effect on tumor growth, stronger than that of anti-EGFR mAb, anti-LAG3 mAb, and anti-EGFR mAb + anti-LAG3 mAb (Figure 8A). The tumor growth inhibition rate was 137.1% in the CMD005 group (10 mg/kg), 27.1% in the anti-EGFR mAb group (10 mg/kg), 86.2% in the anti-LAG3 mAb group (10 mg/kg), and 60.2% in the anti-EGFR mAb + LAG3 mAb group (10 + 10 mg/kg). There were only minor changes in body weight in all groups of mice (Figure 8B).

In summary, in vivo studies have shown that EGFR×LAG3 BsAb CMD005 has a long serum half-life and can specifically and effectively inhibit tumor growth. Therefore, this bispecific antibody therapy, EGFR×LAG3 BsAb CMD005, has great potential for clinical development.

While preferred embodiments of the invention have been shown and described herein, such embodiments will be apparent to those skilled in the art only as examples. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The appended claims are intended to define the scope of the invention and to cover the methods and structures within the scope of these claims and their equivalents.

Invention Field

This application relates to bispecific antibodies targeting EGFR and LAG3, and the uses of such antibodies, particularly their use in cancer treatment.

Background Technology

Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. It consists of a kinase-active C-terminal intracellular region, an N-terminal extracellular receptor binding site, and a hydrophobic transmembrane domain. EGFR is a receptor for tyrosine kinases (RTKs). RTKs play a crucial role in controlling fundamental cellular processes such as cell circulation, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. EGFR can regulate a wide range of cellular processes via tyrosine kinase-regulated signal transduction pathways. Furthermore, EGFR activation leads to the simultaneous activation of multiple signal transduction cascades, including the MAPK pathway, the protein kinase C (PKC) pathway, and the PI(3)K-activated AKT pathway.

EGFR is frequently overexpressed or mutated in human tumors and contributes to tumor growth. Approximately 30% of solid tumors show gain-of-function genetic alterations in EGFR, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. Some cells exhibit "proto-oncogene addiction," a concept introduced by Weinstein in 2002, because they rely on the EGFR signaling pathway for survival and growth. Therefore, targeting EGFR could be a very effective treatment approach. Various strategies targeting EGFR and blocking the EGFR signaling pathway have been reported, including cetuximab and panitumumab. All of these strategies confirm that targeting EGFR is an effective anti-tumor therapy.

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered in an experiment designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines. It is a 70 kDa transmembrane protein structurally homologous to CD4 with four extracellular glycosylation sites. LAG3 is not expressed in resting peripheral blood lymphocytes but is expressed in activated T cells, NK cells, some B cells, and dendritic cells. Like CD4, LAG3 binds to MHC class II molecules with high affinity. However, unlike CD4, LAG3 does not interact with the human immunodeficiency virus gp120 protein. Studies using soluble LAG3 immunoglobulin fusion protein (sLAG3Ig) have shown that LAG3 binds directly and specifically to MHC class II molecules on the cell surface.

The cytoplasmic tail of LAG3 consists of three conserved motifs, one of which is a highly unique and conserved six-amino acid sequence (KIEELE, SEQ ID NO:20) that has been shown to be essential for LAG3-mediated T cell function downregulation. The interaction of LAG3 with its ligands (MHC class II molecules expressed on antigen-presenting cells, such as macrophages and dendritic cells) inhibits the activation of T cells and NK cells, suppressing their ability to recognize and kill cancer cells. Furthermore, CD4 ⁺ CD25 ⁺ regulatory T cells (T _reg ) have been shown to express LAG3 upon activation, and antibodies against LAG3 have inhibited the inhibition induced by induced T _reg cells both in vitro and in vivo, suggesting that LAG3 contributes to the repressor activity of T _reg cells.

The roles of EGFR and LAG3 in cancer are well-established, and while monotherapy targeting either EGFR or LAG3 may initially be highly effective in cancer treatment, resistance often develops. Therefore, there is a need for bispecific antibodies that target both EGFR and LAG3, which are predicted to achieve effective cancer treatment by recognizing both antigens simultaneously.

Invention Overview

This disclosure provides recombinant EGFR×LAG3 bispecific antibodies designed to bind simultaneously to surface antigens on target cells and certain immune cells, such as T cells. Numerous functional assays have demonstrated the potent antitumor effects of antibodies engineered in the form of EGFR×LAG3 bispecific antibodies against a variety of cancers, particularly EGFR-positive cancers such as colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer.

In one aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises the amino acid sequence shown in SEQ ID NO:4; VH1 comprises the amino acid sequence shown in SEQ ID NO:8; VL2 comprises the amino acid sequence shown in SEQ ID NO:12; and VH2 comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from the N-terminus to the C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from the N-terminus to the C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some embodiments, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2 and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2 and IgG4 (e.g., human IgG1, IgG2 and IgG4).

In some implementations, CL is derived from the λ light chain or the κ light chain.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5, and preferably the linker comprises an amino acid sequence as shown in SEQ ID NO:19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:18.

In another aspect, this disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

In another aspect, this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

In another respect, this disclosure provides pharmaceutical compositions comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides conjugates comprising the bispecific antibody or its antigen-binding fragment disclosed herein, and the chemical portion conjugated thereto.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In another aspect, this disclosure provides a method of treating a subject with cancer, including administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject's cancer.

In another aspect, this disclosure provides for the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

In some embodiments of the uses disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Attached Figure Description

The features and advantages of the invention can be understood by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments utilizing the principles of the invention. In the drawings:

Figure 1 schematically shows the structure of the EGFR×LAG3 bispecific antibody (BsAb) CMD005.

Figure 2A shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human EGFR as measured by ELISA.

Figure 2B shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human LAG3 as measured by ELISA.

Figure 3A shows the binding of EGFR×LAG3 BsAb CMD005 to the EGFR-expressing cell line Caco-2, as measured by flow cytometry.

Figure 3B shows the binding of EGFR×LAG3 BsAb CMD005 to LAG3 on the surface of activated T cells, as measured by flow cytometry.

Figure 4A shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between EGFR and EGF.

Figure 4B shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between LAG3 and FGL1.

Figure 5 shows the ability of EGFR×LAG3 BsAb CMD005, as measured by flow cytometry, to block the interaction between LAG3 and MHC class II molecules expressed in Raji cells.

Figure 6A shows the growth inhibition of A431 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6B shows the growth inhibition of Caco-2 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6C shows the growth inhibition of Achn cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 7 shows the plasma concentrations of EGFR×LAG3 BsAb CMD005 in mice treated with CMD005 at 15, 39, 63, and 87 hours post-treatment.

Figure 8A shows the inhibition of EGFR×LAG3BsAb CMD005 on MC38-hEGFR tumor growth in hLAG3-c57 BL/6 mice by tumor volume measurement.

Figure 8B shows the body weight of hLAG3-c57 BL/6 mice in all groups during the tumor growth inhibition assay.

Invention Details

The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the following detailed description of the embodiments.

The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended to provide a general understanding of the invention. These embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are denoted using similar reference numerals throughout the specification.

Unless otherwise stated or defined, all terms used have the common meaning in the art that is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. (eds.), “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; and Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), vols. 1-3, Cold Spring Harbor Laboratory Press. (1989); F. Ausubel et al., eds., “Current protocols in molecular biology”, Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology” (6th edition), Mosby/Elsevier, Edinburgh (2001); and Janeway et al., “Immunology” (6th edition), Garland Science Publishing/Churchill Livingstone, New York (2005), and the general background techniques cited above.

definition

As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

Unless otherwise stated or defined, the term “comprising” and its variations such as “including” and “containing” should be understood to mean that the said element or step or group of elements or steps is included, but does not exclude any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

The “light chain variable region (VL)” or “heavy chain variable region (VH)” consists of a “framework” region interspersed with three “complementarity-determining regions” or “CDRs”. The framework region is used to modulate the CDRs to specifically bind to antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.

The amino acid sequence of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia Kabat CDR, where CDR-H1 is a complex of Chothia CDR and Kabat CDR; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat.

Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five types (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain.

As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense and includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of one or more antibodies that retains the ability to specifically bind antigens. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody.

Examples of antigen-binding fragments covered by the term "antigen-binding moiety" of antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; (vii) dAb fragments, which consist of a VH domain; (viii) separate complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked using recombination methods through synthetic linkers that enable them to be made into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies should also be covered under the term "antigen-binding fragment" in the term antibody. Additionally, the term also includes "linear antibodies," which comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) forming the antigen-binding region together with a complementary light chain polypeptide, and any modified versions of the aforementioned fragments that retain antigen-binding activity.

These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. Affinity, expressed as the equilibrium constant (KD) for antibody-antigen dissociation, is a measure of the strength of binding between the antigenic determinant and the antibody's antigen-binding site: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1/KD.

Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity involves the affinity between an antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies bind to antigens with a dissociation constant (KD) of ^10⁻⁵ to ^10⁻¹² M or less, preferably ^10⁻⁷ to ^10⁻¹² M or less, more preferably ^10⁻⁸ to ^10⁻¹² M, and/or with a binding affinity of at least ^{10⁷ M⁻¹} , preferably at least ^{10⁸ M⁻¹} , more preferably at least ^{10⁹ M⁻¹} , such as at least ^{10¹² M⁻¹} . Any _KD value greater than ^10⁻⁴ M is generally considered to indicate nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatcherd analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations known per se in the art.

The term "epitope" refers to the site on an antigen where an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically consist of at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antibodies and are therefore specific targets for antibodies or their antigen-binding fragments.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percentage of identity between the amino acid sequence and SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gappedBLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid linked to it.

As used in this article, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutical acceptable” means that the carrier or excipient is compatible with the other components of the composition and does not cause significant toxicity to the recipient, and/or that such carrier or excipient is approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms “treatment,” “management,” etc., refer to the administration of a drug or procedure to achieve an effect. These effects may be preventative in terms of the complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of the partial or complete cure of a disease and/or its symptoms. As used herein, “treatment” may include the treatment of a disease or condition (e.g., cancer) in mammals, particularly humans, and includes: (a) the prevention of the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases that may be related to or caused by the primary disease); (b) the suppression of the disease, i.e., the halting of its development; and (c) the alleviation of the disease, i.e., the remission of the disease. Treatment may refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter such as elimination; alleviation; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of deterioration or decline; or making the endpoint of deterioration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of disease, disease symptoms or disease side effects in a subject.

As used in this article, the term "effective amount" refers to an amount that is sufficient to treat a disease when administered to a subject to treat that disease.

As used herein, the term “subject” means any mammalian subject for whom a diagnosis, treatment, or therapy is intended. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

Bispecific antibodies

This disclosure provides a bispecific antibody and its antigen-binding fragment, comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some implementations, the CDR sequence is defined according to the Kabat numbering system.

When the CDR sequence is defined according to the Kabat numbering system, the VL1 of the antibody disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 1 (QASQDISNYLN), SEQ ID NO: 2 (DASNLET), and SEQ ID NO: 3 (QHFDHLPLA), respectively, and the VH1 of the antibody disclosed herein comprises HCDR1, HCDR2, and H having the amino acid sequences shown in SEQ ID NO: 5 (SGDYYWT), SEQ ID NO: 6 (HIYYSGNTNYNPSLKS), and SEQ ID NO: 7 (DRVTGAFDI), respectively. CDR3; The VL2 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 9 (SGSSSNIGSNAVS), SEQ ID NO: 10 (YDDLLPS) and SEQ ID NO: 11 (AAWDDSLNAFV), respectively, and the VH2 of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 13 (SYGIS), SEQ ID NO: 14 (WISAYNGNTNYAQKLQG) and SEQ ID NO: 15 (DSSSWWVDAFDI), respectively.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 4, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VH1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 8, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VL2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 12, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3. In some embodiments, VH2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 16, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In the context of functional variants, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

In some implementations, insertions, deletions, and/or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

In a preferred embodiment, VL1 contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:8; VL2 contains the amino acid sequence shown in SEQ ID NO:12; and VH2 contains the amino acid sequence shown in SEQ ID NO:16.

The bispecific antibodies disclosed herein may contain an Fc region, which includes the antibody's CH2 and CH3 regions.

The Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one implementation, the Fc region is defective in effector function. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector functions such as ADCC. Such mutations have been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001).

In one embodiment, the Fc region contains a mutation that removes the receptor site for Asn-linked glycosylation or is otherwise manipulated to alter the glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in one specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.

In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC can be optimized using the method described by et al. (1999) Nature Biotech 17:176. In a further embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100:2411.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from N-terminus to C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some implementations, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2, and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4). In a preferred embodiment, each of CH1, CH2, and CH3 is independently derived from IgG1.

In some embodiments, CH1-CH2-CH3 comprises an amino acid sequence as shown in any one of SEQ ID NO:21-23. In a preferred embodiment, CH1-CH2-CH3 comprises an amino acid sequence as shown in SEQ ID NO:21.

In some implementations, CL is derived from the λ light chain or the κ light chain. In a preferred implementation, CL is derived from the κ light chain.

In some embodiments, CL comprises an amino acid sequence as shown in any one of SEQ ID NO:24-25. In a preferred embodiment, CL comprises an amino acid sequence as shown in SEQ ID NO:24.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5. In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGS (SEQ ID NO: 26). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGS (SEQ ID NO: 27). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 19). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 29). In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 17 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR and/or LAG3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 18 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In some embodiments, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, even more preferably 5% to 30%, even more preferably 10% to 25%, even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the insertion, deletion, and/or substitution can be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH3.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitution have been described above.

In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18.

Nucleic acid

This disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

The term "nucleic acid" encompasses single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or any modified form of nucleotides. These modifications include base modifications, such as uridine bromide and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and modifications involving nucleotide linkages, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

In some embodiments, the nucleic acid is a ribonucleotide (RNA) or a deoxyribonucleotide (DNA). In some embodiments, the present invention provides a ribonucleotide (RNA) comprising a nucleotide sequence encoding a bispecific antibody disclosed herein. In some embodiments, the present invention provides a deoxyribonucleotide comprising a deoxynucleotide sequence encoding a bispecific antibody disclosed herein.

In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

In some embodiments, the ribonucleotide (RNA) may be introduced into human cells in vivo. In some embodiments, the ribonucleotide (RNA) of the present invention is contained in a carrier or delivery agent.

carrier

This disclosure provides vectors containing the nucleic acids disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy or light chain variable region of a bispecific antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

Any vector may be suitable for use in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs containing a replication system functional in prokaryotic or eukaryotic host cells can be prepared. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

For example, the vector could be an adenovirus vector containing a nucleotide sequence encoding the bispecific antibody disclosed herein. This vector could be administered to a subject and subsequently enter cells within the subject, thereby integrating the nucleotide sequence encoding the bispecific antibody disclosed herein into the cell's genome, and subsequently, the cell would express the bispecific antibody disclosed herein.

host cells

This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium. The bacteria include *Serratia* (e.g., *Serratia marcescans*), *Shigella*, *Bacilli* (e.g., *B. subtilis* and *B. licheniformis*), *Pseudomonas* (e.g., *P. aeruginosa*), and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or in vivo. The host cells of the present invention can be administered to a subject in vivo, and the host cells express the bispecific antibodies disclosed herein in vivo.

The present invention provides host cells in which any of the above-described vectors have been introduced. The invention further provides a method for preparing the bispecific antibody of the present invention, wherein the method comprises a) culturing the host cells of the fourth aspect of the present invention under conditions suitable for the production of bispecific antibodies; and b) obtaining the bispecific antibody from the culture.

Pharmaceutical Composition

This disclosure provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and pharmaceutically acceptable carriers or excipients.

Bispecific antibodies or their antigen-binding fragments, or the pharmaceutical agents of the present invention (also referred to herein as "active ingredients") and their derivatives, fragments, analogs, and homologs, may be incorporated into pharmaceutical compositions for ease of administration. Such compositions typically comprise a bispecific antibody or its antigen-binding fragment or a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with the use of the drug. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and anhydrous media (such as fixed oils) may also be used. The use of such media and pharmaceutical agents for the active pharmaceutical ingredient is well known in the art. The use of any conventional media or pharmaceutical agent in the composition is considered except where it is incompatible with the active compound. Supplemental active compounds may also be incorporated into the composition.

In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate; and reagents for adjusting pH, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (wherein being water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL ^™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the desired amount of the active compound, along with one or a combination of the ingredients listed above, into a suitable solvent as needed, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of preparing sterile powders for sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce a powder containing the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binder and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavorings.

For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a sprayer.

Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for mucosal application. Mucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion known in the art.

The active compound can also be formulated as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

This invention provides therapeutic compositions comprising the bispecific antibody of the invention or an antigen-binding fragment thereof. The therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in formulations known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN ^™ ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm SciTechnol 52:238-311.

Conjugate

This disclosure provides conjugates comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the chemical moiety conjugated thereto.

In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as “immunoconjugates.” When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an “antibody-drug conjugate” or “ADC.”

The term "conjugation" or "linkage" refers to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

The chemical moiety can be linked to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent linkages can be used. The procedure for linking the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to generate binding to the chemical moiety. Alternatively, the antibody may be derivatized to expose or link additional reactive functional groups. Derivatization may involve linking any of a number of known adapter molecules. The adapter can be any molecule used to link the antibody to the chemical moiety. The adapter is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable adapters are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon adapters, heterocyclic carbon adapters, or peptide adapters. In the case where the antibody and chemical moiety are peptides, the adapter can be linked via their side groups to the constituent amino acids (e.g., via a disulfide bond with cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

In some cases, when an immunoconjugate reaches its target site, it is desirable to release the chemical portion from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.

Conditions experienced by enzymatically active or immunoconjugates within or near target cells may induce linker cleavage to release the chemical portion from the antibody.

Given the numerous methods already reported for linking various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art will be able to determine suitable methods for linking a given reagent to an antibody or other peptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally linked (through chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a dimeric antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (such as the streptavidin core region or a multihistidine tag).

One type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types). Suitable cross-linking agents include heterobifunctional cross-linking agents having two distinctly reactive groups separated by a suitable spacer (such as m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional cross-linking agents (such as disuccinimide octanoate). Such linkers are commercially available.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be, for example, maytansine, galdromycin, a microtubule inhibitor such as a microtubule binding agent (e.g., olistatin), or a minor groove binding agent such as chaziomycin.

Other suitable therapeutic agents include small molecule cytotoxic agents, which are compounds with a molecular weight of less than 700 Daltons capable of killing mammalian cells. These compounds may also contain toxic metals that produce cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, which are compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, chalcogenide, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porphyrin sodium photosensitizer II, temozolomide, topotecan, trimesartan gluconate, olprestatin E, vincristine, and doxorubicin; and peptide cytotoxic agents, which are proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, Dnase, and RNase; radionuclides, which are unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying. Examples include iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents can be used to promote the binding of these radionuclides to molecules or polymers thereof.

In some embodiments, the detectable portion may be selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Detectable portions for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

Bispecific antibodies can be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance transduction (MTR), ultrasound, fiber optic examination, and laparoscopy. Specifically, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used.

Bispecific antibodies or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a bispecific antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding an additional reagent that typically produces a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Bispecific antibodies or antigen-binding fragments can also be conjugated to biotin and detected indirectly by binding to avidin or streptavidin. It should be noted that avidin itself can bind to enzymes or fluorescent labels.

Bispecific antibodies can be fused with self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned to the ends of constant regions. HaloTag is a self-labeled protein tag derived from bacterial enzymes (haloalkane dehalogenases) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligands contain chloroalkane linkers attached to fluorophores such as near-infrared fluorophores (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

Bispecific antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanide elements (such as europium and dysprosium) and manganese.

Paramagnetic particles, such as superparamagnetic iron oxide, can also be used as labels. Bispecific antibodies can also be labeled with predetermined peptide epitopes recognized by a second reporter molecule (such as leucine zipper pairs, binding sites of the second antibody, metal-binding domains, or epitope tags). In some embodiments, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

Bispecific antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, and 131I.

In some implementations, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine, such as IL-2 and IFN-γ; a chemokine, such as IL-8; platelet-4; melanoma growth-stimulating protein; a complement activator; a viral/bacterial protein domain; or a viral/bacterial peptide.

Treatment

This disclosure provides a method for treating a subject with cancer, comprising administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer.

Examples of cancer include: leukemia, such as, but not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and myelodysplastic syndromes); chronic leukemia, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; polycythemia vera; lymphoma, such as, but not limited to, Hodgkin's disease and non-Hodgkin's disease; and multiple myeloma, such as, but not limited to... Smoking multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; unexplained monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, and soft tissue sarcoma. Angiosarcoma, (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, glioblastoma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pinealoblastoma, primary brain lymphoma; breast cancer, This includes, but is not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid carcinoma, medullary thyroid adenoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma; GIST-gastrointestinal stromal tumors; and pancreatic cancer, such as, but not limited to, islet tumors, gastrinomas, glucagonomas, vasodilator intestinal peptide tumors, and somatostatin-secreting tumors. Cancers include: ovarian tumors and carcinoid tumors or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas like iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancers. Cancers, such as but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancers, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancers, such as but not limited to adenocarcinoma, mycosis fungoides, ulcerative, superficially spreading, widely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancers, such as but not limited to hepatocellular carcinoma and hepatoblastic carcinoma; gallbladder cancers, such as but not limited to adenocarcinoma; bile duct cancers, such as but not limited to breast cancers. Head-shaped, nodular, and diffuse cholangiocarcinoma; lung cancer, such as non-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer (SCLC); testicular cancer, such as, but not limited to, germ cell tumors, seminoma, anaplastic, classic (typical), spermatogenic cell tumors, non-seminomatous tumors, embryonal carcinoma, teratoma, and choriocarcinoma (yolk sac tumor); prostate cancer, such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers, such as penile cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as, but not limited to, Limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and warts; skin cancer, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigines melanoma, and acral lentigines melanoma; renal cancer, such as, but not limited to, renal cell carcinoma, clear cell renal cell carcinoma, adenocarcinoma, adrenoidoma, fibrosarcoma, and transitional cell carcinoma (renal pelvis and/or ureter); Wilms' tumor; bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioma endothelial sarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. Preferably, the cancer is selected from breast cancer, melanoma, prostate cancer, ovarian cancer, colorectal cancer, lung cancer, or glioma.

In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some implementations, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment can depend on many factors, such as antibody activity and the route of administration.

A single dose of the antibody, composition, or conjugate described herein may be administered to mammals once or in a series of sub-dose over appropriate time periods, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the antibody, composition, or conjugate may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more sub-dose administrations per day.

The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, antibodies, compositions, or conjugates are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the antibody, composition, or conjugate disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

Medical Use

This disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject’s cancer.

This disclosure also provides the disclosed bispecific antibodies or antigen-binding fragments thereof, the disclosed pharmaceutical compositions, or the disclosed conjugates for use in treating cancer in subjects.

In some embodiments of the uses disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Reagent test kit

This disclosure provides pharmaceutical kits or reagent kits comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as the bispecific antibodies or antigen-binding fragments disclosed herein. Optionally, associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for manufacture, use, or sale for human administration.

In one embodiment, the kit includes a first container of the bispecific antibody disclosed herein. In another embodiment, the kit includes a first container which is a vial containing the bispecific antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

In one embodiment, this document provides an injection device comprising a bispecific antibody. In one embodiment, the injection device comprises a bispecific antibody in a sterile solution. In another embodiment, the injection device is a syringe.

Example

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, are representative of preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Variations and other uses covered by the spirit of the invention as defined by the claims will be apparent to those skilled in the art.

MC38-hEGFR cancer cells were purchased from Kyinno Biotechnology Co., Ltd. Activated T cells were obtained by activating frozen PBMCs with human CD3/CD28 T cell activators. Other tumor cell lines, including human epidermal cancer cell line A431, human lymphoma cell line Raji, human colon cancer cell line Caco-2, and renal cancer cell line Achn, were purchased from ATCC.

Human EGF protein and human FGL1 protein were purchased from ACROBiosystems. Human EGFR/HER1/ErbB1 protein and human LAG3 protein were purchased from Sino Biological. Anti-human IgG (γ chain specific)-R-PE antibody, anti-human IgG (Fc specific)-peroxidase antibody, and monoclonal anti-peroxidase were purchased from Sigma. PE anti-His tag antibody was purchased from BioLegend. Anti-EGFR mAb and anti-LAG3 mAb were prepared in-house.

The full light chain and heavy chain sequences of the Anti-EGFR mAb and Anti-LAG3 mAb are shown below.

Anti-EGFR mAb

Heavy chain:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34) light chain:

DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:35)

Anti-LAG3 mAb

Heavy chain:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDSSSWWVDAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:36)

Light chain:

SYELTQPPSVSEAPRQRVTISSCSGSSSNIGSNAVSWYQQVPRKAPKLLVYYDDLLPSGVSDRFSGSKSGTSASLAIRGLQSEDEADYYCAAWDDSLNAFVFGTGTKVTVLGQPK ANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:37)

Example 1. Construction and preliminary characterization of EGFR×LAG3 bispecific antibody

Cloning of EGFR×LAG3 bispecific antibody (BsAb)

The light chain of the EGFR×LAG3 BsAb consists of an anti-LAG-3VL and a CL domain. The heavy chain, from the N-terminus to the C-terminus, contains an anti-LAG-3VH, a CH1 domain, a wild-type human IgG1 Fc, and an anti-EGFR single-chain variable region fragment (ScFv). The structure of the EGFR×LAG3 BsAb (also referred to herein as CMD005) is shown in Figure 1. The light chain of both the anti-EGFR ScFv and the EGFR×LAG3 BsAb was synthesized directly by GENEWIZ. The following primers, synthesized by GENEWIZ, were used to generate the EGFR×LAG3 BsAb construct:

Vector-HC-FP:5’TAATTCTAGAGTCGACAATCAACCTCTGG 3’(Justice) (SEQ ID NO:30);

Vector-HC-RP:5’TGATCCACCACCTCCACTACCG 3’(antense) (SEQ ID NO:31);

EGFR-HB-pa-VH-FP:

5’GTGGTGGATCACAGGTGCAGCTGCAGGAGAG 3’(Justice) (SEQ ID NO:32);

EGFR-HB-pa-VL-RP:

5’GATTGTCGACTCTAGAATTATTTGATTTCCACCTTGGTTCCG3’ (antisense) (SEQ ID NO:33).

To generate EGFR×LAG3 BsAb, anti-EGFR ScFv was fused to the C-terminus of the LAG-3 monoclonal antibody via a (G4S)4 linker. The light and heavy chains were separately constructed into a single vector pcDNA3.4 for mammalian cell expression.

Protein expression, purification and preliminary characterization

EGFR×LAG3 BsAb was expressed in CHO-S cells using the ExpiFectamine CHO transfection kit. Cells continued to grow for 5–7 days post-transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The supernatant containing the target protein was loaded onto a gravity column equipped with EshmunoA (Merck). Subsequent purification was performed according to the manufacturer's instructions.

EGFR×LAG3 BsAb was well expressed in transiently transfected CHO-S cells and secreted into the culture supernatant. On non-reducing SDS-PAGE, the apparent molecular weight (aMW) of CMD005 was approximately 76.3 kDa. On reduced SDS-PAGE, the heavy and light chains were close together, with an apparent molecular weight of approximately 23.1 kDa (data not shown).

The CDR sequence of CMD005 according to the Kabat numbering system is shown in Table 1. The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of CMD005 are shown in Table 2. The complete light chain and heavy chain sequences of CMD005 are shown in Table 3.

Table 1. CDR Sequence of CMD005

Table 2. VL and VH sequences of CMD005

Table 3. Heavy and light chain sequences of CMD005

Example 2. Binding of EGFR×LAG3 BsAb CMD005 with recombinant EGFR and LAG3

ELISA was performed according to standard protocol to determine the binding affinity of CMD005 for recombinant EGFR and LAG3. Briefly, human antigens EGFR or LAG3 were coated at 100 ng per well overnight at 4°C on Corning EIA/RIA high-binding 96-well plates (Corning Inc.), and blocked with PBS (pH 7.4, MPBS) containing 3% skim milk, 3% bovine serum albumin, and 1‰ Tween 20, respectively. Antibodies were added in 5-fold serial dilutions and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected using HRP-conjugated streptavidin (Sino Biological). This assay was developed using TMB substrates (Solarbio) at room temperature and monitored using a microplate reader at 450 nm. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1 kappa (IgG1κ) isotype control was used as a negative control. The results are shown in Figures 2A-2B.

The results showed that CMD005 could bind to recombinant human EGFR with an EC50 of 0.43 nM and recombinant human LAG3 with an EC50 of 0.09 nM. Furthermore, there was no significant difference in binding activity between the EGFR×LAG3 bispecific antibody CMD005 and anti-EGFR or anti-LAG3 mAbs.

Example 3: EGFR×LAG3 BsAb CMD005 binding to cell surface-associated EGFR and LAG3

To measure the binding ability of EGFR×LAG3 BsAb CMD005 to LAG3-associated cells on the cell surface, flow cytometry was performed using activated T cells, and the binding ability of CMD005 to EGFR-associated cells on the cell surface was measured by flow cytometry using the human colon cancer cell line Caco-2. Approximately 5 × ^10⁵ cells were incubated on ice for 1 hour with five-fold serially diluted antibody. Cells were washed once with PBS containing 0.5% bovine serum albumin (PBSA) and resuspended in 100 μL of PBSA. Then, 1 μL of anti-human IgG (γ-chain specific)-R-phycoerythrin antibody ((Sigma)) generated in goats was added and incubated for 30 minutes. Cells were washed once with PBSA and then used for flow cytometry analysis. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 3A-3B.

The results showed that EGFR×LAG3 BsAb CMD005 could bind well to Caco-2 and T cells, and the binding affinity was similar to that of anti-EGFR mAb or anti-LAG3 mAb.

Example 4. Blocking of EGFR-EGF, LAG3-FGL1, and LAG3-MHCII binding mediated by EGFR×LAG3 BsAb CMD005

ELISA assays were performed to determine the ability of EGFR×LAG3 BsAb CMD005 to block the binding of EGFR/EGF and LAG3/FGL1 receptor-ligand pairs.

Human antigen EGFR was coated at 500 ng per well on Corning EIA/RIA high-binding 96-well plates (Corning Inc.) at 4 °C overnight and blocked with 3% MPBS (pH 7.4). Five serially diluted antibodies were mixed with human antigen EGF (14 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected by HRP anti-6X antibody (Abcam). This assay was developed using TMB substrate (Solarbio) at room temperature and monitored at 450 nm using a microplate reader. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1κ isotype control was used as a negative control. Results are shown in Figure 4A.

Human antigen FGL1 was coated at 100 ng per well on plates overnight at 4°C and blocked with PBS containing 3% bovine serum albumin and 1‰ Tween 20. Five serially diluted antibodies were mixed with human antigen LAG3 (4 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The remaining steps were the same as above. Human IgG1κ isotype control was used as a negative control. The results are shown in Figure 4B.

The blocking ability of CMD005 against the LAG3/MHCII receptor-ligand pair was measured using cell-based competitive flow cytometry. Approximately 5 × ^10⁵ Raji cells expressing MHCII on their cell surface were incubated on ice for 1 h with serially diluted antibody solution and biotin-labeled human antigen LAG3 (400 ng). Cells were washed once with 0.5% PBSA and then resuspended in 100 μL of PBSA. Then, 0.1% streptavidin R-PE conjugate (Invitrogen) was added and the cells were incubated on ice for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. A human IgG1κ isotype control was used as a negative control. The results are shown in Figure 5.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong blocking effect on the receptor-ligand interaction of EGFR/EGF and LAG3/FGL1, with EC50 values of 1.86 nM and 32.85 nM, respectively (Figures 4A-4B). Furthermore, CMD005 inhibited or competed with MHCII for binding to LAG3, with an EC50 value of 6.88 nM (Figure 5).

Example 5. EGFR×LAG3 BsAb CMD005-mediated killing of human cancer cell lines

The in vitro efficacy of EGFR×LAG3 BsAb CMD005 was evaluated using several cancer cell lines, including A431, Caco-2, and Achn, via CCK8 assay. These three cell lines were centrifuged and resuspended in RPMI 1640 complete medium, then added to 96-well round-bottom plates at densities of 1000, 4000, and 1500 cells/100 μL, respectively. Cells were incubated at 37°C with 5% _CO2 supplementation for 24 hours. The next day, 100 μL of antibody solution (serially diluted 10-fold from 100 nM) was added to each well accordingly. After 4–10 days of incubation (cell confluence 80–90%), the medium was removed from the target cells, and 100 μL of RPMI 1640 complete medium containing 10% CCK-8 was added, followed by incubation in a _CO2 incubator for 30 minutes. Cell inhibitory activity was measured using a microplate reader according to the manufacturer's instructions. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 6A-6C.

The results showed that EGFR×LAG3 BsAb CMD005 exhibited potent inhibition of tumor cell growth in a dose-dependent manner. There was no significant difference in the inhibitory effect of EGFR×LAG3 BsAb CMD005 on tumor cell growth compared to anti-EGFR mAb.

Example 6. Inhibition of tumor growth in mice mediated by anti-AXL bispecific antibody

Measurement of pharmacokinetics in mice

On day 0, two BALB/c mice were intravenously administered 200 μg of EGFR×LAG3 BsAb CMD005. Plasma samples were collected at 15, 39, 63, and 87 hours post-treatment for measuring serum antibody concentrations by ELISA, and a standard curve was generated using the original antibody stock. The results are shown in Figure 7.

The results showed that the serum concentration of CMD005 gradually decreased, but remained at a high level at the endpoint.

In vivo tumor growth inhibition

The in vivo efficacy of EGFR×LAG3 BsAb CMD005 was evaluated in hLAG3-c57 BL/6 mice. Three × ^10⁶ MC38-hEGFR tumor cells were subcutaneously seeded into the right abdomen of hLAG3-c57 BL/6 mice. After identifying palpable tumors, mice were randomized according to tumor volume and body weight. A negative control group was administered 10 mg/kg of human IgG1κ isotype intravenously. Experimental groups included an anti-EGFR mAb group (10 mg/kg), an anti-LAG3 mAb group (10 mg/kg), an anti-EGFR mAb + anti-LAG3 mAb group (10 + 10 mg/kg), and a CMD005 group (10 mg/kg). These mice were administered the drugs twice weekly. After three weeks of treatment, the mice were sacrificed and tumor volume was measured. The tumor growth inhibition (TGI) rate was calculated using the following formula: TGI(%) = [1 - ( _TT0 )/( _CC0 )] × 100 (T and _T0 : tumor volumes on the specific experimental day and the randomized experimental day, respectively; C and _C0 : the corresponding average tumor volumes in the control group). A value > 100% indicates tumor shrinkage. The results are shown in Figures 8A-8B.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong inhibitory effect on tumor growth, stronger than that of anti-EGFR mAb, anti-LAG3 mAb, and anti-EGFR mAb + anti-LAG3 mAb (Figure 8A). The tumor growth inhibition rate was 137.1% in the CMD005 group (10 mg/kg), 27.1% in the anti-EGFR mAb group (10 mg/kg), 86.2% in the anti-LAG3 mAb group (10 mg/kg), and 60.2% in the anti-EGFR mAb + LAG3 mAb group (10 + 10 mg/kg). There were only minor changes in body weight in all groups of mice (Figure 8B).

In summary, in vivo studies have shown that EGFR×LAG3 BsAb CMD005 has a long serum half-life and can specifically and effectively inhibit tumor growth. Therefore, this bispecific antibody therapy, EGFR×LAG3 BsAb CMD005, has great potential for clinical development.

While preferred embodiments of the invention have been shown and described herein, such embodiments will be apparent to those skilled in the art only as examples. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The appended claims are intended to define the scope of the invention and to cover the methods and structures within the scope of these claims and their equivalents.

Bispecific antibodies targeting both EGFR and LAG3 and their applications

Invention Field

This application relates to bispecific antibodies targeting EGFR and LAG3, and the uses of such antibodies, particularly their use in cancer treatment.

Background Technology

Epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. It consists of a kinase-active C-terminal intracellular region, an N-terminal extracellular receptor binding site, and a hydrophobic transmembrane domain. EGFR is a receptor for tyrosine kinases (RTKs). RTKs play a crucial role in controlling fundamental cellular processes such as cell circulation, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. EGFR can regulate a wide range of cellular processes via tyrosine kinase-regulated signal transduction pathways. Furthermore, EGFR activation leads to the simultaneous activation of multiple signal transduction cascades, including the MAPK pathway, the protein kinase C (PKC) pathway, and the PI(3)K-activated AKT pathway.

EGFR is frequently overexpressed or mutated in human tumors and contributes to tumor growth. Approximately 30% of solid tumors show gain-of-function genetic alterations in EGFR, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. Some cells exhibit "proto-oncogene addiction," a concept introduced by Weinstein in 2002, because they rely on the EGFR signaling pathway for survival and growth. Therefore, targeting EGFR could be a very effective treatment approach. Various strategies targeting EGFR and blocking the EGFR signaling pathway have been reported, including cetuximab and panitumumab. All of these strategies confirm that targeting EGFR is an effective anti-tumor therapy.

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered in an experiment designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines. It is a 70 kDa transmembrane protein structurally homologous to CD4 with four extracellular glycosylation sites. LAG3 is not expressed in resting peripheral blood lymphocytes but is expressed in activated T cells, NK cells, some B cells, and dendritic cells. Like CD4, LAG3 binds to MHC class II molecules with high affinity. However, unlike CD4, LAG3 does not interact with the human immunodeficiency virus gp120 protein. Studies using soluble LAG3 immunoglobulin fusion protein (sLAG3Ig) have shown that LAG3 binds directly and specifically to MHC class II molecules on the cell surface.

The cytoplasmic tail of LAG3 consists of three conserved motifs, one of which is a highly unique and conserved six-amino acid sequence (KIEELE, SEQ ID NO:20) that has been shown to be essential for LAG3-mediated T cell function downregulation. The interaction of LAG3 with its ligands (MHC class II molecules expressed on antigen-presenting cells, such as macrophages and dendritic cells) inhibits the activation of T cells and NK cells, suppressing their ability to recognize and kill cancer cells. Furthermore, CD4 ⁺ CD25 ⁺ regulatory T cells (T _reg ) have been shown to express LAG3 upon activation, and antibodies against LAG3 have inhibited the inhibition induced by induced T _reg cells both in vitro and in vivo, suggesting that LAG3 contributes to the repressor activity of T _reg cells.

The roles of EGFR and LAG3 in cancer are well-established, and while monotherapy targeting either EGFR or LAG3 may initially be highly effective in cancer treatment, resistance often develops. Therefore, there is a need for bispecific antibodies that target both EGFR and LAG3, which are predicted to achieve effective cancer treatment by recognizing both antigens simultaneously.

Invention Overview

This disclosure provides recombinant EGFR×LAG3 bispecific antibodies designed to bind simultaneously to surface antigens on target cells and certain immune cells, such as T cells. Numerous functional assays have demonstrated the potent antitumor effects of antibodies engineered in the form of EGFR×LAG3 bispecific antibodies against a variety of cancers, particularly EGFR-positive cancers such as colon cancer, kidney cancer, colorectal cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer.

In one aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises the amino acid sequence shown in SEQ ID NO:4; VH1 comprises the amino acid sequence shown in SEQ ID NO:8; VL2 comprises the amino acid sequence shown in SEQ ID NO:12; and VH2 comprises the amino acid sequence shown in SEQ ID NO:16.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from the N-terminus to the C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from the N-terminus to the C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some embodiments, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2 and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2 and IgG4 (e.g., human IgG1, IgG2 and IgG4).

In some implementations, CL is derived from the λ light chain or the κ light chain.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5, and preferably the linker comprises an amino acid sequence as shown in SEQ ID NO:19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:18.

In another aspect, this disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

In another aspect, this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

In another respect, this disclosure provides pharmaceutical compositions comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides conjugates comprising the bispecific antibody or its antigen-binding fragment disclosed herein, and the chemical portion conjugated thereto.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In another aspect, this disclosure provides a method of treating a subject with cancer, including administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

On the other hand, this disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject's cancer.

In another aspect, this disclosure provides for the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

In some embodiments of the uses disclosed herein, the cancer is EGFR-positive. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Attached Figure Description

The features and advantages of the invention can be understood by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments utilizing the principles of the invention. In the drawings:

Figure 1 schematically shows the structure of the EGFR×LAG3 bispecific antibody (BsAb) CMD005.

Figure 2A shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human EGFR as measured by ELISA.

Figure 2B shows the binding of EGFR×LAG3 BsAb CMD005 to recombinant human LAG3 as measured by ELISA.

Figure 3A shows the binding of EGFR×LAG3 BsAb CMD005 to the EGFR-expressing cell line Caco-2, as measured by flow cytometry.

Figure 3B shows the binding of EGFR×LAG3 BsAb CMD005 to LAG3 on the surface of activated T cells, as measured by flow cytometry.

Figure 4A shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between EGFR and EGF.

Figure 4B shows the ability of EGFR×LAG3 BsAb CMD005, as measured by ELISA, to block the interaction between LAG3 and FGL1.

Figure 5 shows the ability of EGFR×LAG3 BsAb CMD005, as measured by flow cytometry, to block the interaction between LAG3 and MHC class II molecules expressed in Raji cells.

Figure 6A shows the growth inhibition of A431 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6B shows the growth inhibition of Caco-2 cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 6C shows the growth inhibition of Achn cells by EGFR×LAG3 BsAb CMD005 in vitro.

Figure 7 shows the plasma concentrations of EGFR×LAG3 BsAb CMD005 in mice treated with CMD005 at 15, 39, 63, and 87 hours post-treatment.

Figure 8A shows the inhibition of EGFR×LAG3BsAb CMD005 on MC38-hEGFR tumor growth in hLAG3-c57 BL/6 mice by tumor volume measurement.

Figure 8B shows the body weight of hLAG3-c57 BL/6 mice in all groups during the tumor growth inhibition assay.

Invention Details

The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below by taking into account the accompanying drawings and the following detailed description of the embodiments.

The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended to provide a general understanding of the invention. These embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are denoted using similar reference numerals throughout the specification.

Unless otherwise stated or defined, all terms used have the common meaning in the art that is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. (eds.), “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; and Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), vols. 1-3, Cold Spring Harbor Laboratory Press. (1989); F. Ausubel et al., eds., “Current protocols in molecular biology”, Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology” (6th edition), Mosby/Elsevier, Edinburgh (2001); and Janeway et al., “Immunology” (6th edition), Garland Science Publishing/Churchill Livingstone, New York (2005), and the general background techniques cited above.

definition

As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

Unless otherwise stated or defined, the term “comprising” and its variations such as “including” and “containing” should be understood to mean that the said element or step or group of elements or steps is included, but does not exclude any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

The “light chain variable region (VL)” or “heavy chain variable region (VH)” consists of a “framework” region interspersed with three “complementarity-determining regions” or “CDRs”. The framework region is used to modulate the CDRs to specifically bind to antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.

The amino acid sequence of each VL and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia Kabat CDR, where CDR-H1 is a complex of Chothia CDR and Kabat CDR; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat.

Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five types (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain.

As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. As used herein, the term "bispecific antibody" should be understood in its broadest sense and includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of one or more antibodies that retains the ability to specifically bind antigens. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody.

Examples of antigen-binding fragments covered by the term "antigen-binding moiety" of antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; (vii) dAb fragments, which consist of a VH domain; (viii) separate complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked using recombination methods through synthetic linkers that enable them to be made into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies should also be covered under the term "antigen-binding fragment" in the term antibody. Additionally, the term also includes "linear antibodies," which comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) forming the antigen-binding region together with a complementary light chain polypeptide, and any modified versions of the aforementioned fragments that retain antigen-binding activity.

These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. Affinity, expressed as the equilibrium constant (KD) for antibody-antigen dissociation, is a measure of the strength of binding between the antigenic determinant and the antibody's antigen-binding site: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1/KD.

Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity involves the affinity between an antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies bind to antigens with a dissociation constant (KD) of ^10⁻⁵ to ^10⁻¹² M or less, preferably ^10⁻⁷ to ^10⁻¹² M or less, more preferably ^10⁻⁸ to ^10⁻¹² M, and/or with a binding affinity of at least ^{10⁷ M⁻¹} , preferably at least ^{10⁸ M⁻¹} , more preferably at least ^{10⁹ M⁻¹} , such as at least ^{10¹² M⁻¹} . Any _KD value greater than ^10⁻⁴ M is generally considered to indicate nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, Scatcherd analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations known per se in the art.

The term "epitope" refers to the site on an antigen where an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically consist of at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antibodies and are therefore specific targets for antibodies or their antigen-binding fragments.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percentage of identity between the amino acid sequence and SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gappedBLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

As used in this article, the term "carrier" refers to a nucleic acid molecule that can transport another nucleic acid linked to it.

As used in this article, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutical acceptable” means that the carrier or excipient is compatible with the other components of the composition and does not cause significant toxicity to the recipient, and/or that such carrier or excipient is approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms “treatment,” “management,” etc., refer to the administration of a drug or procedure to achieve an effect. These effects may be preventative in terms of the complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of the partial or complete cure of a disease and/or its symptoms. As used herein, “treatment” may include the treatment of a disease or condition (e.g., cancer) in mammals, particularly humans, and includes: (a) the prevention of the occurrence of a disease or its symptoms in subjects susceptible to the disease but not yet diagnosed with it (e.g., including diseases that may be related to or caused by the primary disease); (b) the suppression of the disease, i.e., the halting of its development; and (c) the alleviation of the disease, i.e., the remission of the disease. Treatment may refer to any indication of success in treating or improving or preventing cancer, including any objective or subjective parameter such as elimination; alleviation; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of deterioration or decline; or making the endpoint of deterioration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of disease, disease symptoms or disease side effects in a subject.

As used in this article, the term "effective amount" refers to an amount that is sufficient to treat a disease when administered to a subject to treat that disease.

As used herein, the term “subject” means any mammalian subject for whom a diagnosis, treatment, or therapy is intended. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sporting animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

Bispecific antibodies

This disclosure provides a bispecific antibody and its antigen-binding fragment, comprising a first antigen-binding region binding to EGFR, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and a second antigen-binding region binding to LAG3, the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:1-3; VH1 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:5-7; VL2 comprises LCDR1-3 having amino acid sequences as shown in SEQ ID NO:9-11; and VH2 comprises HCDR1-3 having amino acid sequences as shown in SEQ ID NO:13-15.

In some implementations, the CDR sequence is defined according to the Kabat numbering system.

When the CDR sequence is defined according to the Kabat numbering system, the VL1 of the antibody disclosed herein comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 1 (QASQDISNYLN), SEQ ID NO: 2 (DASNLET), and SEQ ID NO: 3 (QHFDHLPLA), respectively, and the VH1 of the antibody disclosed herein comprises HCDR1, HCDR2, and H having the amino acid sequences shown in SEQ ID NO: 5 (SGDYYWT), SEQ ID NO: 6 (HIYYSGNTNYNPSLKS), and SEQ ID NO: 7 (DRVTGAFDI), respectively. CDR3; The VL2 of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO: 9 (SGSSSNIGSNAVS), SEQ ID NO: 10 (YDDLLPS) and SEQ ID NO: 11 (AAWDDSLNAFV), respectively, and the VH2 of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 13 (SYGIS), SEQ ID NO: 14 (WISAYNGNTNYAQKLQG) and SEQ ID NO: 15 (DSSSWWVDAFDI), respectively.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4; VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

In some embodiments, VL1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 4, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VH1 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 8, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR. In some embodiments, VL2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 12, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3. In some embodiments, VH2 comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 16, formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In the context of functional variants, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

In some implementations, insertions, deletions, and/or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

The particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

In a preferred embodiment, VL1 contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:8; VL2 contains the amino acid sequence shown in SEQ ID NO:12; and VH2 contains the amino acid sequence shown in SEQ ID NO:16.

The bispecific antibodies disclosed herein may contain an Fc region, which includes the antibody's CH2 and CH3 regions.

The Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one implementation, the Fc region is defective in effector function. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector functions such as ADCC. Such mutations have been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001).

In one embodiment, the Fc region contains a mutation that removes the receptor site for Asn-linked glycosylation or is otherwise manipulated to alter the glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in one specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.

In a further embodiment, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC can be optimized using the method described by et al. (1999) Nature Biotech 17:176. In a further embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100:2411.

In some embodiments, the bispecific antibody comprises: a first polypeptide chain comprising, from N-terminus to C-terminus: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and a second polypeptide chain comprising, from N-terminus to C-terminus: VL2-CL; wherein CH1 represents a first domain of the constant region of the immunoglobulin heavy chain; CH2 represents a second domain of the constant region of the immunoglobulin heavy chain; CH3 represents a third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

In some implementations, the bispecific antibody comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain.

In some embodiments, each of CH1, CH2, and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably from an IgG subtype selected from IgG1, IgG2, and IgG4 (e.g., human IgG1, IgG2, and IgG4). In a preferred embodiment, each of CH1, CH2, and CH3 is independently derived from IgG1.

In some embodiments, CH1-CH2-CH3 comprises an amino acid sequence as shown in any one of SEQ ID NO:21-23. In a preferred embodiment, CH1-CH2-CH3 comprises an amino acid sequence as shown in SEQ ID NO:21.

In some implementations, CL is derived from the λ light chain or the κ light chain. In a preferred implementation, CL is derived from the κ light chain.

In some embodiments, CL comprises an amino acid sequence as shown in any one of SEQ ID NO:24-25. In a preferred embodiment, CL comprises an amino acid sequence as shown in SEQ ID NO:24.

In some embodiments, the linker comprises an amino acid sequence of (G4S)n, where n is an integer selected from 1 to 5. In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGS (SEQ ID NO: 26). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGS (SEQ ID NO: 27). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 19). In some embodiments, the linker may comprise an amino acid sequence as shown in GGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 29). In a preferred embodiment, the linker comprises an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and the second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

In some embodiments, the first polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 17 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to EGFR and/or LAG3. In some embodiments, the second polypeptide chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 18 formed by inserting, deleting, and/or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to LAG3.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

In some embodiments, the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33% of the total number of amino acids in the parent amino acid sequence, even more preferably 5% to 30%, even more preferably 10% to 25%, even more preferably 15% to 20%. For example, the number of inserted, deleted, and/or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the insertion, deletion, and/or substitution can be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, such as CL, CH1, CH2, and/or CH3.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitution have been described above.

In a preferred embodiment, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 17; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18.

Nucleic acid

This disclosure provides nucleic acids comprising nucleotide sequences encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein.

The term "nucleic acid" encompasses single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or any modified form of nucleotides. These modifications include base modifications, such as uridine bromide and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and modifications involving nucleotide linkages, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding: (i) any of the heavy chain variable region sequences disclosed herein and (ii) any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

For example, the present invention provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding: (i) a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

In some embodiments, the nucleic acid is a ribonucleotide (RNA) or a deoxyribonucleotide (DNA). In some embodiments, the present invention provides a ribonucleotide (RNA) comprising a nucleotide sequence encoding a bispecific antibody disclosed herein. In some embodiments, the present invention provides a deoxyribonucleotide comprising a deoxynucleotide sequence encoding a bispecific antibody disclosed herein.

In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

In some embodiments, the ribonucleotide (RNA) may be introduced into human cells in vivo. In some embodiments, the ribonucleotide (RNA) of the present invention is contained in a carrier or delivery agent.

carrier

This disclosure provides vectors containing the nucleic acids disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy or light chain variable region of a bispecific antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

Any vector may be suitable for use in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs containing a replication system functional in prokaryotic or eukaryotic host cells can be prepared. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

For example, the vector could be an adenovirus vector containing a nucleotide sequence encoding the bispecific antibody disclosed herein. This vector could be administered to a subject and subsequently enter cells within the subject, thereby integrating the nucleotide sequence encoding the bispecific antibody disclosed herein into the cell's genome, and subsequently, the cell would express the bispecific antibody disclosed herein.

host cells

This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium. The bacteria include *Serratia* (e.g., *Serratia marcescans*), *Shigella*, *Bacilli* (e.g., *B. subtilis* and *B. licheniformis*), *Pseudomonas* (e.g., *P. aeruginosa*), and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or in vivo. The host cells of the present invention can be administered to a subject in vivo, and the host cells express the bispecific antibodies disclosed herein in vivo.

The present invention provides host cells in which any of the above-described vectors have been introduced. The invention further provides a method for preparing the bispecific antibody of the present invention, wherein the method comprises a) culturing the host cells of the fourth aspect of the present invention under conditions suitable for the production of bispecific antibodies; and b) obtaining the bispecific antibody from the culture.

Pharmaceutical Composition

This disclosure provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and pharmaceutically acceptable carriers or excipients.

Bispecific antibodies or their antigen-binding fragments, or the pharmaceutical agents of the present invention (also referred to herein as "active ingredients") and their derivatives, fragments, analogs, and homologs, may be incorporated into pharmaceutical compositions for ease of administration. Such compositions typically comprise a bispecific antibody or its antigen-binding fragment or a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with the use of the drug. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and anhydrous media (such as fixed oils) may also be used. The use of such media and pharmaceutical agents for the active pharmaceutical ingredient is well known in the art. The use of any conventional media or pharmaceutical agent in the composition is considered except where it is incompatible with the active compound. Supplemental active compounds may also be incorporated into the composition.

In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate; and reagents for adjusting pH, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (wherein being water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL ^™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersed state, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the desired amount of the active compound, along with one or a combination of the ingredients listed above, into a suitable solvent as needed, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of preparing sterile powders for sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce a powder containing the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binder and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavorings.

For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a sprayer.

Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for mucosal application. Mucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion known in the art.

The active compound can also be formulated as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

This invention provides therapeutic compositions comprising the bispecific antibody of the invention or an antigen-binding fragment thereof. The therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in formulations known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN ^™ ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), J Pharm SciTechnol 52:238-311.

Conjugate

This disclosure provides conjugates comprising the bispecific antibody or antigen-binding fragment thereof disclosed herein, and the chemical moiety conjugated thereto.

In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as “immunoconjugates.” When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an “antibody-drug conjugate” or “ADC.”

The term "conjugation" or "linkage" refers to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

The chemical moiety can be linked to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent linkages can be used. The procedure for linking the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to generate binding to the chemical moiety. Alternatively, the antibody may be derivatized to expose or link additional reactive functional groups. Derivatization may involve linking any of a number of known adapter molecules. The adapter can be any molecule used to link the antibody to the chemical moiety. The adapter is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable adapters are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon adapters, heterocyclic carbon adapters, or peptide adapters. In the case where the antibody and chemical moiety are peptides, the adapter can be linked via their side groups to the constituent amino acids (e.g., via a disulfide bond with cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

In some cases, when an immunoconjugate reaches its target site, it is desirable to release the chemical portion from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.

Conditions experienced by enzymatically active or immunoconjugates within or near target cells may induce linker cleavage to release the chemical portion from the antibody.

Given the numerous methods already reported for linking various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art will be able to determine suitable methods for linking a given reagent to an antibody or other peptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally linked (through chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a dimeric antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (such as the streptavidin core region or a multihistidine tag).

One type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types). Suitable cross-linking agents include heterobifunctional cross-linking agents having two distinctly reactive groups separated by a suitable spacer (such as m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional cross-linking agents (such as disuccinimide octanoate). Such linkers are commercially available.

In some embodiments of the conjugates disclosed herein, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be, for example, maytansine, galdromycin, a microtubule inhibitor such as a microtubule binding agent (e.g., olistatin), or a minor groove binding agent such as chaziomycin.

Other suitable therapeutic agents include small molecule cytotoxic agents, which are compounds with a molecular weight of less than 700 Daltons capable of killing mammalian cells. These compounds may also contain toxic metals that produce cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, which are compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, chalcogenide, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porphyrin sodium photosensitizer II, temozolomide, topotecan, trimesartan gluconate, olprestatin E, vincristine, and doxorubicin; and peptide cytotoxic agents, which are proteins or fragments thereof capable of killing mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, Dnase, and RNase; radionuclides, which are unstable isotopes of elements that emit one or more alpha or beta particles or gamma rays while decaying. Examples include iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213; chelating agents can be used to promote the binding of these radionuclides to molecules or polymers thereof.

In some embodiments, the detectable portion may be selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules. Detectable portions for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

Bispecific antibodies can be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance transduction (MTR), ultrasound, fiber optic examination, and laparoscopy. Specifically, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used.

Bispecific antibodies or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When a bispecific antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding an additional reagent that typically produces a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Bispecific antibodies or antigen-binding fragments can also be conjugated to biotin and detected indirectly by binding to avidin or streptavidin. It should be noted that avidin itself can bind to enzymes or fluorescent labels.

Bispecific antibodies can be fused with self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned to the ends of constant regions. HaloTag is a self-labeled protein tag derived from bacterial enzymes (haloalkane dehalogenases) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligands contain chloroalkane linkers attached to fluorophores such as near-infrared fluorophores (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

Bispecific antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanide elements (such as europium and dysprosium) and manganese.

Paramagnetic particles, such as superparamagnetic iron oxide, can also be used as labels. Bispecific antibodies can also be labeled with predetermined peptide epitopes recognized by a second reporter molecule (such as leucine zipper pairs, binding sites of the second antibody, metal-binding domains, or epitope tags). In some embodiments, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

Bispecific antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, and 131I.

In some implementations, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule may be a cytokine, such as IL-2 and IFN-γ; a chemokine, such as IL-8; platelet-4; melanoma growth-stimulating protein; a complement activator; a viral/bacterial protein domain; or a viral/bacterial peptide.

Treatment

This disclosure provides a method for treating a subject with cancer, comprising administering to the subject an effective amount of the bispecific antibody disclosed herein or an antigen-binding fragment thereof, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer.

Examples of cancer include: leukemia, such as, but not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and myelodysplastic syndromes); chronic leukemia, such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; polycythemia vera; lymphoma, such as, but not limited to, Hodgkin's disease and non-Hodgkin's disease; and multiple myeloma, such as, but not limited to... Smoking multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; unexplained monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, and soft tissue sarcoma. Angiosarcoma, (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, glioblastoma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pinealoblastoma, primary brain lymphoma; breast cancer, This includes, but is not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast carcinoma, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid carcinoma, medullary thyroid adenoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma; GIST-gastrointestinal stromal tumors; and pancreatic cancer, such as, but not limited to, islet tumors, gastrinomas, glucagonomas, vasodilator intestinal peptide tumors, and somatostatin-secreting tumors. Cancers include: ovarian tumors and carcinoid tumors or islet cell tumors; pituitary cancers, such as, but not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancers, such as, but not limited to, ocular melanomas like iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancers such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers, such as, but not limited to, endometrial cancer and uterine sarcoma; ovarian cancers. Cancers, such as but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancers, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancers, such as but not limited to adenocarcinoma, mycosis fungoides, ulcerative, superficially spreading, widely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancers, such as but not limited to hepatocellular carcinoma and hepatoblastic carcinoma; gallbladder cancers, such as but not limited to adenocarcinoma; bile duct cancers, such as but not limited to breast cancers. Head-shaped, nodular, and diffuse cholangiocarcinoma; lung cancer, such as non-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer (SCLC); testicular cancer, such as, but not limited to, germ cell tumors, seminoma, anaplastic, classic (typical), spermatogenic cell tumors, non-seminomatous tumors, embryonal carcinoma, teratoma, and choriocarcinoma (yolk sac tumor); prostate cancer, such as, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers, such as penile cancer; oral cancer, such as, but not limited to, squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as, but not limited to, Limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as, but not limited to, squamous cell carcinoma and warts; skin cancer, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigines melanoma, and acral lentigines melanoma; renal cancer, such as, but not limited to, renal cell carcinoma, clear cell renal cell carcinoma, adenocarcinoma, adrenoidoma, fibrosarcoma, and transitional cell carcinoma (renal pelvis and/or ureter); Wilms' tumor; bladder cancer, such as, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioma endothelial sarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. Preferably, the cancer is selected from breast cancer, melanoma, prostate cancer, ovarian cancer, colorectal cancer, lung cancer, or glioma.

In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some implementations, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site and subject being treated. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment can depend on many factors, such as antibody activity and the route of administration.

A single dose of the antibody, composition, or conjugate described herein may be administered to mammals once or in a series of sub-dose over appropriate time periods, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the antibody, composition, or conjugate may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more sub-dose administrations per day.

The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, antibodies, compositions, or conjugates are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

In some embodiments, the method further includes administering a second therapeutic agent to the subject. In some embodiments, the antibody, composition, or conjugate disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapy agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracene diones, anthracyclines, podophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cefpaclitaxel, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimine, nitrogen mustard, nitrosourea, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

Medical Use

This disclosure provides the use of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject’s cancer.

This disclosure also provides the disclosed bispecific antibodies or antigen-binding fragments thereof, the disclosed pharmaceutical compositions, or the disclosed conjugates for use in treating cancer in subjects.

In some embodiments of the uses disclosed herein, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is EGFR-positive cancer. In some embodiments, the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer. In a preferred embodiment, the cancer is selected from colorectal cancer, head and neck cancer, kidney cancer, and skin cancer.

In some embodiments, the bispecific antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is combined with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

Reagent test kit

This disclosure provides pharmaceutical kits or reagent kits comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as the bispecific antibodies or antigen-binding fragments disclosed herein. Optionally, associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for manufacture, use, or sale for human administration.

In one embodiment, the kit includes a first container of the bispecific antibody disclosed herein. In another embodiment, the kit includes a first container which is a vial containing the bispecific antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

In one embodiment, this document provides an injection device comprising a bispecific antibody. In one embodiment, the injection device comprises a bispecific antibody in a sterile solution. In another embodiment, the injection device is a syringe.

Example

The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, are representative of preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Variations and other uses covered by the spirit of the invention as defined by the claims will be apparent to those skilled in the art.

MC38-hEGFR cancer cells were purchased from Kyinno Biotechnology Co., Ltd. Activated T cells were obtained by activating frozen PBMCs with human CD3/CD28 T cell activators. Other tumor cell lines, including human epidermal cancer cell line A431, human lymphoma cell line Raji, human colon cancer cell line Caco-2, and renal cancer cell line Achn, were purchased from ATCC.

Human EGF protein and human FGL1 protein were purchased from ACROBiosystems. Human EGFR/HER1/ErbB1 protein and human LAG3 protein were purchased from Sino Biological. Anti-human IgG (γ chain specific)-R-PE antibody, anti-human IgG (Fc specific)-peroxidase antibody, and monoclonal anti-peroxidase were purchased from Sigma. PE anti-His tag antibody was purchased from BioLegend. Anti-EGFR mAb and anti-LAG3 mAb were prepared in-house.

The full light chain and heavy chain sequences of the Anti-EGFR mAb and Anti-LAG3 mAb are shown below.

Anti-EGFR mAb

Heavy chain:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34) light chain:

DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:35)

Anti-LAG3 mAb

Heavy chain:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDSSSWWVDAFDIWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKKVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:36)

Light chain:

SYELTQPPSVSEAPRQRVTISSCSGSSSNIGSNAVSWYQQVPRKAPKLLVYYDDLLPSGVSDRFSGSKSGTSASLAIRGLQSEDEADYYCAAWDDSLNAFVFGTGTKVTVLGQPK ANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:37)

Example 1. Construction and preliminary characterization of EGFR×LAG3 bispecific antibody

Cloning of EGFR×LAG3 bispecific antibody (BsAb)

The light chain of the EGFR×LAG3 BsAb consists of an anti-LAG-3VL and a CL domain. The heavy chain, from the N-terminus to the C-terminus, contains an anti-LAG-3VH, a CH1 domain, a wild-type human IgG1 Fc, and an anti-EGFR single-chain variable region fragment (ScFv). The structure of the EGFR×LAG3 BsAb (also referred to herein as CMD005) is shown in Figure 1. The light chain of both the anti-EGFR ScFv and the EGFR×LAG3 BsAb was synthesized directly by GENEWIZ. The following primers, synthesized by GENEWIZ, were used to generate the EGFR×LAG3 BsAb construct:

Vector-HC-FP:5’TAATTCTAGAGTCGACAATCAACCTCTGG 3’(Justice) (SEQ ID NO:30);

Vector-HC-RP:5’TGATCCACCACCTCCACTACCG 3’(antense) (SEQ ID NO:31);

EGFR-HB-pa-VH-FP:

5’GTGGTGGATCACAGGTGCAGCTGCAGGAGAG 3’(Justice) (SEQ ID NO:32);

EGFR-HB-pa-VL-RP:

5’GATTGTCGACTCTAGAATTATTTGATTTCCACCTTGGTTCCG3’ (antisense) (SEQ ID NO:33).

To generate EGFR×LAG3 BsAb, anti-EGFR ScFv was fused to the C-terminus of the LAG-3 monoclonal antibody via a (G4S)4 linker. The light and heavy chains were separately constructed into a single vector pcDNA3.4 for mammalian cell expression.

Protein expression, purification and preliminary characterization

EGFR×LAG3 BsAb was expressed in CHO-S cells using the ExpiFectamine CHO transfection kit. Cells continued to grow for 5–7 days post-transfection. Cell cultures were harvested by centrifugation at 8000 rpm for 20 minutes. The supernatant containing the target protein was loaded onto a gravity column equipped with EshmunoA (Merck). Subsequent purification was performed according to the manufacturer's instructions.

EGFR×LAG3 BsAb was well expressed in transiently transfected CHO-S cells and secreted into the culture supernatant. On non-reducing SDS-PAGE, the apparent molecular weight (aMW) of CMD005 was approximately 76.3 kDa. On reduced SDS-PAGE, the heavy and light chains were close together, with an apparent molecular weight of approximately 23.1 kDa (data not shown).

The CDR sequence of CMD005 according to the Kabat numbering system is shown in Table 1. The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of CMD005 are shown in Table 2. The complete light chain and heavy chain sequences of CMD005 are shown in Table 3.

Table 1. CDR Sequence of CMD005

Table 2. VL and VH sequences of CMD005

Table 3. Heavy and light chain sequences of CMD005

Example 2. Binding of EGFR×LAG3 BsAb CMD005 with recombinant EGFR and LAG3

ELISA was performed according to standard protocol to determine the binding affinity of CMD005 for recombinant EGFR and LAG3. Briefly, human antigens EGFR or LAG3 were coated at 100 ng per well overnight at 4°C on Corning EIA/RIA high-binding 96-well plates (Corning Inc.), and blocked with PBS (pH 7.4, MPBS) containing 3% skim milk, 3% bovine serum albumin, and 1‰ Tween 20, respectively. Antibodies were added in 5-fold serial dilutions and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected using HRP-conjugated streptavidin (Sino Biological). This assay was developed using TMB substrates (Solarbio) at room temperature and monitored using a microplate reader at 450 nm. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1 kappa (IgG1κ) isotype control was used as a negative control. The results are shown in Figures 2A-2B.

The results showed that CMD005 could bind to recombinant human EGFR with an EC50 of 0.43 nM and recombinant human LAG3 with an EC50 of 0.09 nM. Furthermore, there was no significant difference in binding activity between the EGFR×LAG3 bispecific antibody CMD005 and anti-EGFR or anti-LAG3 mAbs.

Example 3: EGFR×LAG3 BsAb CMD005 binding to cell surface-associated EGFR and LAG3

To measure the binding ability of EGFR×LAG3 BsAb CMD005 to LAG3-associated cells on the cell surface, flow cytometry was performed using activated T cells, and the binding ability of CMD005 to EGFR-associated cells on the cell surface was measured by flow cytometry using the human colon cancer cell line Caco-2. Approximately 5 × ^10⁵ cells were incubated on ice for 1 hour with five-fold serially diluted antibody. Cells were washed once with PBS containing 0.5% bovine serum albumin (PBSA) and resuspended in 100 μL of PBSA. Then, 1 μL of anti-human IgG (γ-chain specific)-R-phycoerythrin antibody ((Sigma)) generated in goats was added and incubated for 30 minutes. Cells were washed once with PBSA and then used for flow cytometry analysis. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 3A-3B.

The results showed that EGFR×LAG3 BsAb CMD005 could bind well to Caco-2 and T cells, and the binding affinity was similar to that of anti-EGFR mAb or anti-LAG3 mAb.

Example 4. Blocking of EGFR-EGF, LAG3-FGL1, and LAG3-MHCII binding mediated by EGFR×LAG3 BsAb CMD005

ELISA assays were performed to determine the ability of EGFR×LAG3 BsAb CMD005 to block the binding of EGFR/EGF and LAG3/FGL1 receptor-ligand pairs.

Human antigen EGFR was coated at 500 ng per well on Corning EIA/RIA high-binding 96-well plates (Corning Inc.) at 4 °C overnight and blocked with 3% MPBS (pH 7.4). Five serially diluted antibodies were mixed with human antigen EGF (14 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The plates were washed with PBS containing 0.05% Tween 20. Binding antibodies were detected by HRP anti-6X antibody (Abcam). This assay was developed using TMB substrate (Solarbio) at room temperature and monitored at 450 nm using a microplate reader. The half-maximal binding ( _EC50 ) was calculated by fitting the data to Langmuir adsorption isotherms. Human IgG1κ isotype control was used as a negative control. Results are shown in Figure 4A.

Human antigen FGL1 was coated at 100 ng per well on plates overnight at 4°C and blocked with PBS containing 3% bovine serum albumin and 1‰ Tween 20. Five serially diluted antibodies were mixed with human antigen LAG3 (4 μg/mL) in an equal volume ratio. The mixture was added to the plates and incubated at room temperature for 2 hours. The remaining steps were the same as above. Human IgG1κ isotype control was used as a negative control. The results are shown in Figure 4B.

The blocking ability of CMD005 against the LAG3/MHCII receptor-ligand pair was measured using cell-based competitive flow cytometry. Approximately 5 × ^10⁵ Raji cells expressing MHCII on their cell surface were incubated on ice for 1 h with serially diluted antibody solution and biotin-labeled human antigen LAG3 (400 ng). Cells were washed once with 0.5% PBSA and then resuspended in 100 μL of PBSA. Then, 0.1% streptavidin R-PE conjugate (Invitrogen) was added and the cells were incubated on ice for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. A human IgG1κ isotype control was used as a negative control. The results are shown in Figure 5.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong blocking effect on the receptor-ligand interaction of EGFR/EGF and LAG3/FGL1, with EC50 values of 1.86 nM and 32.85 nM, respectively (Figures 4A-4B). Furthermore, CMD005 inhibited or competed with MHCII for binding to LAG3, with an EC50 value of 6.88 nM (Figure 5).

Example 5. EGFR×LAG3 BsAb CMD005-mediated killing of human cancer cell lines

The in vitro efficacy of EGFR×LAG3 BsAb CMD005 was evaluated using several cancer cell lines, including A431, Caco-2, and Achn, via CCK8 assay. These three cell lines were centrifuged and resuspended in RPMI 1640 complete medium, then added to 96-well round-bottom plates at densities of 1000, 4000, and 1500 cells/100 μL, respectively. Cells were incubated at 37°C with 5% _CO2 supplementation for 24 hours. The next day, 100 μL of antibody solution (serially diluted 10-fold from 100 nM) was added to each well accordingly. After 4–10 days of incubation (cell confluence 80–90%), the medium was removed from the target cells, and 100 μL of RPMI 1640 complete medium containing 10% CCK-8 was added, followed by incubation in a _CO2 incubator for 30 minutes. Cell inhibitory activity was measured using a microplate reader according to the manufacturer's instructions. Human IgG1κ isotype control was used as a negative control. The results are shown in Figures 6A-6C.

The results showed that EGFR×LAG3 BsAb CMD005 exhibited potent inhibition of tumor cell growth in a dose-dependent manner. There was no significant difference in the inhibitory effect of EGFR×LAG3 BsAb CMD005 on tumor cell growth compared to anti-EGFR mAb.

Example 6. Inhibition of tumor growth in mice mediated by anti-AXL bispecific antibody

Measurement of pharmacokinetics in mice

On day 0, two BALB/c mice were intravenously administered 200 μg of EGFR×LAG3 BsAb CMD005. Plasma samples were collected at 15, 39, 63, and 87 hours post-treatment for measuring serum antibody concentrations by ELISA, and a standard curve was generated using the original antibody stock. The results are shown in Figure 7.

The results showed that the serum concentration of CMD005 gradually decreased, but remained at a high level at the endpoint.

In vivo tumor growth inhibition

The in vivo efficacy of EGFR×LAG3 BsAb CMD005 was evaluated in hLAG3-c57 BL/6 mice. Three × ^10⁶ MC38-hEGFR tumor cells were subcutaneously seeded into the right abdomen of hLAG3-c57 BL/6 mice. After identifying palpable tumors, mice were randomized according to tumor volume and body weight. A negative control group was administered 10 mg/kg of human IgG1κ isotype intravenously. Experimental groups included an anti-EGFR mAb group (10 mg/kg), an anti-LAG3 mAb group (10 mg/kg), an anti-EGFR mAb + anti-LAG3 mAb group (10 + 10 mg/kg), and a CMD005 group (10 mg/kg). These mice were administered the drugs twice weekly. After three weeks of treatment, the mice were sacrificed and tumor volume was measured. The tumor growth inhibition (TGI) rate was calculated using the following formula: TGI(%) = [1 - ( _TT0 )/( _CC0 )] × 100 (T and _T0 : tumor volumes on the specific experimental day and the randomized experimental day, respectively; C and _C0 : the corresponding average tumor volumes in the control group). A value > 100% indicates tumor shrinkage. The results are shown in Figures 8A-8B.

The results showed that EGFR×LAG3 BsAb CMD005 had a strong inhibitory effect on tumor growth, stronger than that of anti-EGFR mAb, anti-LAG3 mAb, and anti-EGFR mAb + anti-LAG3 mAb (Figure 8A). The tumor growth inhibition rate was 137.1% in the CMD005 group (10 mg/kg), 27.1% in the anti-EGFR mAb group (10 mg/kg), 86.2% in the anti-LAG3 mAb group (10 mg/kg), and 60.2% in the anti-EGFR mAb + LAG3 mAb group (10 + 10 mg/kg). There were only minor changes in body weight in all groups of mice (Figure 8B).

In summary, in vivo studies have shown that EGFR×LAG3 BsAb CMD005 has a long serum half-life and can specifically and effectively inhibit tumor growth. Therefore, this bispecific antibody therapy, EGFR×LAG3 BsAb CMD005, has great potential for clinical development.

While preferred embodiments of the invention have been shown and described herein, such embodiments will be apparent to those skilled in the art only as examples. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The appended claims are intended to define the scope of the invention and to cover the methods and structures within the scope of these claims and their equivalents.

Claims (25)

1. A bispecific antibody or its antigen-binding fragment thereof, said bispecific antibody or its antigen-binding fragment comprising a first antigen-binding region binding to EGFR and a second antigen-binding region binding to LAG3, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein

The VL1 comprises LCDR1-3, each having an amino acid sequence as shown in SEQ ID NO:1-3;

The VH1 comprises HCDR1-3, each having an amino acid sequence as shown in SEQ ID NO:5-7;

The VL2 comprises LCDR1-3, each having an amino acid sequence as shown in SEQ ID NO: 9-11; and

The VH2 comprises HCDR1-3, each having an amino acid sequence as shown in SEQ ID NO:13-15.

2. The bispecific antibody or its antibody-binding fragment according to claim 1, wherein...

The VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:4;

The VH1 contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8;

The VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:12; and

The VH2 contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:16.

3. The bispecific antibody or its antibody-binding fragment according to claim 2, wherein...

The VL1 contains the amino acid sequence shown in SEQ ID NO:4;

The VH1 contains an amino acid sequence as shown in SEQ ID NO:8;

The VL2 contains the amino acid sequence shown in SEQ ID NO:12; and

The VH2 contains an amino acid sequence as shown in SEQ ID NO:16.

4. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the bispecific antibody comprises:

The first polypeptide chain, from its N-terminus to its C-terminus, comprises: VH2-CH1-CH2-CH3-L1-VH1-L2-VL1; and

The second polypeptide chain, from the N-terminus to the C-terminus, contains: VL2-CL;

Wherein CH1 represents the first domain of the constant region of the immunoglobulin heavy chain; CH2 represents the second domain of the constant region of the immunoglobulin heavy chain; CH3 represents the third domain of the constant region of the immunoglobulin heavy chain; CL represents the constant region of the immunoglobulin light chain; and L1 and L2 each independently represent optional linkers.

5. The bispecific antibody or its antigen-binding fragment according to claim 4, wherein the bispecific antibody comprises two copies of the first polypeptide chain and two copies of the second polypeptide chain.

6. The bispecific antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein each of CH1, CH2 and CH3 is independently derived from an immunoglobulin isotype IgG (e.g., human IgG), preferably derived from an IgG subtype selected from IgG1, IgG2 and IgG4 (e.g., human IgG1, IgG2 and IgG4).

7. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 4-6, wherein the CL is derived from the λ light chain or the κ light chain.

8. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 4-7, wherein the adapter comprises an amino acid sequence of (G4S)n, wherein n is an integer selected from 1-5, preferably the adapter comprises an amino acid sequence as shown in SEQ ID NO:19.

9. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 4-8, wherein...

The first polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:17; and

The second polypeptide chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.

10. The bispecific antibody or its antigen-binding fragment according to claim 9, wherein...

The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:17; and

The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:18.

11. A nucleic acid comprising a nucleotide sequence encoding a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1-10.

12. A vector comprising the nucleic acid according to claim 11.

13. A host cell comprising the nucleic acid according to claim 11 or the vector according to claim 12.

14. A pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1-10, and a pharmaceutically acceptable carrier or excipient.

15. The pharmaceutical composition of claim 14, further comprising a second therapeutic agent.

16. The pharmaceutical composition of claim 15, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

17. The pharmaceutical composition of claim 15 or 16, wherein the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.

18. A conjugate comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1-10, and a chemical moiety conjugated thereto.

19. The conjugate of claim 18, wherein the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

20. A method of treating a subject with cancer, comprising administering to the subject an effective amount of a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1-10, a pharmaceutical composition according to any one of claims 14-17, or a conjugate according to claim 18 or 19.

21. The method of claim 20, wherein the cancer is an EGFR-positive cancer.

22. The method according to claim 21, wherein the cancer is selected from colon cancer, kidney cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, brain cancer, bladder cancer, and liver cancer, preferably colorectal cancer, head and neck cancer, kidney cancer, or skin cancer.

23. The method according to any one of claims 20-22, further comprising administering a second therapeutic agent to the subject.

24. The method of claim 23, wherein the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

25. The method of claim 23 or 24, wherein the second therapeutic agent is selected from Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1/PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, VEGF inhibitors, and glucocorticoids.