Trispecific antibodies and uses thereof

Abstract

Disclosed herein are trispecific antibodies comprising a first antigen-binding region that binds to PD1, a second antigen-binding region that binds to CTLA4, and a third antigen-binding region that binds to VEGFa. Also disclosed are nucleic acids encoding the trispecific antibodies, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors, as well as medical uses of the trispecific antibodies.

Description

[0001] This application claims priority to PCT applications filed on September 30, 2024, with application number PCT / CN2024 / 122851, and PCT / CN2025 / 119744, filed on September 8, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to trispecific antibodies that bind to PD1, CTLA-4 and VEGF, and their uses. Background Technology

[0003] Angiogenesis, the development of new blood vessels from pre-existing blood vessels, is crucial for tumor growth and metastasis. Inhibiting angiogenesis presents a potentially valuable strategy for treating diseases such as cancer that rely on new blood vessel formation for progression (e.g., metastasis). Two important angiogenic pathways include the vascular endothelial growth factor (VEGF) pathway and the Tie2 pathway, with the major VEGF pathway mediated by the transmembrane tyrosine kinase VEGF-R2. Various isoforms of VEGF, particularly VEGFa, bind to VEGF-R2, leading to phosphorylation of various downstream tyrosine kinases, resulting in dimerization and activation.

[0004] Besides the role of angiogenesis in cancer progression, the inability of the host to clear cancer cells is another major problem. Although the immune system is the primary mechanism for cancer prevention, cancer cells can counteract immune surveillance. Immune checkpoints PD1 and CTLA-4 exhibit different expression patterns on different immune cells, and their mediated signaling interactions enhance anti-tumor immunity. Therefore, simultaneous blockade of PD1 and CTLA-4 can promote anti-tumor immunity through multiple mechanisms. Bispecific antibodies targeting PD1 and CTLA-4 induce PD1 endocytosis and degradation. In cells that co-express PD1 and CTLA-4 (such as exhausted T cells in the tumor microenvironment), blocking the enhanced interaction between CTLA-4 and its ligand CD80, along with other mechanisms, promotes anti-tumor immunity, demonstrating efficacy in patients who do not respond to immune checkpoint inhibitors in clinical practice (AlexeyBerezhnoy et al., Cell Rep Med. 2020 Dee 22; 1(9): 100163. Dovedi SJ et al., Cancer Discovery, 08 Jan 2021, 11(5):1100-1117).

[0005] Multispecific antibodies that simultaneously target multiple targets hold great promise for the clinical treatment of complex diseases. However, it is recognized in the art that simply linking two or more antibodies or proteins together often does not elicit a synergistic effect and may even have adverse consequences. Trispecific antibodies that can effectively bind to targets and inhibit tumor growth are highly anticipated in this field. Summary of the Invention

[0006] In one aspect, this disclosure provides a trispecific antibody comprising a first antigen-binding region that binds to PD1, a second antigen-binding region that binds to CTLA4, and a third antigen-binding region that binds to VEGFa.

[0007] In some embodiments, the first antigen-binding region comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein: the first VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and the first VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 10, 11 and 12, respectively.

[0008] In some embodiments, the first VH 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: 13, and the first VL 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: 14.

[0009] In some embodiments, the second antigen-binding region comprises a second VH and a second VL. In some embodiments, the third antigen-binding region comprises a third VH and a third VL.

[0010] In some embodiments, the first, second, and third antigen-binding regions are independently in the form of scfv, Fab, Fab, Fab', F(ab')2, scFab, Fv, dsFv, scFv, or di-scFv. In some embodiments, the first, second, and third antigen-binding regions are independently in the form of scfv or Fab. In some embodiments, the first and second antigen-binding regions are in the form of scFv, and the third antigen-binding region is in the form of Fab. In some embodiments, the first and third antigen-binding regions are in the form of scFv, and the second antigen-binding region is in the form of Fab. In some embodiments, the second and third antigen-binding regions are in the form of scFv, and the first antigen-binding region is in the form of Fab.

[0011] In some preferred embodiments, the first antigen-binding region is in the form of an scFv. In some preferred embodiments, the second antigen-binding region is in the form of an scFv. In some preferred embodiments, the third antigen-binding region is in the form of a Fab.

[0012] In some embodiments, the first, second, and third antigen-binding regions are independently monovalent or bivalent. In some embodiments, at least one of the first, second, and third antigen-binding regions is monovalent. In some embodiments, at least one of the first, second, and third antigen-binding regions is bivalent. In some embodiments, the first and second antigen-binding regions are monovalent, and the third antigen-binding region is bivalent. In some embodiments, the first and third antigen-binding regions are monovalent, and the second antigen-binding region is bivalent. In some embodiments, the second and third antigen-binding regions are monovalent, and the first antigen-binding region is bivalent.

[0013] In some preferred embodiments, the first antigen-binding region and the second antigen-binding region are monovalent. In some embodiments, the third antigen-binding region is bivalent.

[0014] In some implementations, the trispecific antibody comprises two heavy chains and two light chains, wherein the two heavy chains comprise heavy chain 1 and heavy chain 2, heavy chain 1 comprises a first antigen-binding region in the form of scFv at its C-terminus, and heavy chain 2 comprises a second antigen-binding region in the form of scFv at its C-terminus.

[0015] In some implementations, each of the two heavy chains contains a third antigen-binding region (VH) at its N-terminus, and each of the two light chains contains a third antigen-binding region (VL) at its N-terminus.

[0016] In some embodiments, the trispecific antibody further includes a CH3 constant region. In some preferred embodiments, the trispecific antibody further includes an Fc region.

[0017] In some implementations, the Fc region has an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

[0018] In some implementations, the Fc region has a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0019] In some implementations, the CH3 constant region contains a knock-in-hole mutation.

[0020] In some embodiments, the second antigen-binding region comprises a second VH and a second VL, wherein the second VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 15, 16 and 17, respectively, and the second VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 18, 19 and 20, respectively.

[0021] In some embodiments, the second VH 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: 21, and the second VL 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: 22.

[0022] In some embodiments, the third antigen-binding region comprises a third VH and a third VL, wherein the third VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 23, 24 and 25, respectively, and the third VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 26, 27 and 28, respectively.

[0023] In some embodiments, the third VH 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: 29, and the third VL 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: 30.

[0024] In some implementations, from the N-terminus to the C-terminus, heavy chain 1 includes: a VH, CH1, and Fc region of the third antigen-binding region and a first antigen-binding region in the form of scFv; heavy chain 2 includes: a VH, CH1, and Fc region of the third antigen-binding region and a second antigen-binding region in the form of scFv; and each of the two light chains includes: a VL region of the third binding region and a light chain constant region.

[0025] In some embodiments, heavy chain 1 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: 32; heavy chain 2 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: 33; and each of the two light chains 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: 31.

[0026] On the other hand, this disclosure provides nucleic acids encoding the trispecific antibodies disclosed herein.

[0027] On the other hand, this disclosure provides vectors containing the nucleic acids disclosed herein.

[0028] On the other hand, this disclosure provides a host cell containing the nucleic acids disclosed herein.

[0029] In another respect, this disclosure provides pharmaceutical compositions comprising the trispecific antibodies disclosed herein, and optionally at least one pharmaceutically acceptable carrier or excipient.

[0030] In some embodiments, the composition further comprises a second therapeutic agent, optionally selected from the group consisting of free antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, peptides, and small molecule drugs.

[0031] On the other hand, this disclosure provides methods for treating a disease, comprising administering to a subject an effective amount of the disclosed trispecific antibody or the disclosed pharmaceutical composition. In some embodiments, the disease is cancer.

[0032] In some embodiments, the cancer is a PD-L1-expressing cancer. In some embodiments, the cancer is a PD-L1-overexpressing cancer. In some embodiments, the disease is PD-L1-negative. In some preferred embodiments, the cancer is a solid tumor.

[0033] In some implementations, solid tumors include, but are not limited to, colon cancer, adenocarcinoma of the rectum and colon, prostate cancer, breast cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), liver cancer (including hepatocellular carcinoma), stomach cancer, renal cancer, kidney cancer (including renal cell carcinoma), cervical cancer, ovarian cancer, and endometrial cancer. Attached Figure Description

[0034] The features and advantages of the invention can be understood by referring to the detailed description of the illustrative embodiments (in which the principles of the invention are utilized) set forth below, along with the accompanying drawings, wherein:

[0035] Figure 1 The structure of J30 is shown.

[0036] Figure 2 The multi-target binding of J30, as detected by Octet, is shown. The time periods indicated by “TAs” correspond to the association of the test sample with VEGFa.

[0037] Figure 3 The binding and ligand blocking effects of J30 or the CS1002+CS1003 combination on PD1 or CTLA4 single-positive and double-positive cells were demonstrated.

[0038] Figure 4 The blocking effect of J30 on the binding of VEGFa and VEGFR2 was demonstrated.

[0039] Figure 5 J30 was shown to inhibit VEGFa-induced proliferation of human umbilical vein endothelial cells.

[0040] Figure 6 The study showed that J30 induces internalization of the target on CHOS-PD1 / CTLA4 cells.

[0041] Figure 7 J30 was shown to inhibit the growth of MC38-PDL1 tumor cells in humanized PD1 / PDL1 / CTLA4 mice.

[0042] Figure 8 The median tumor volume and Kaplan-Meier survival curves are shown in CTLA4 / VEGFR2 mice with MC38-VEGFA tumor cells.

[0043] Figure 9 This study demonstrates the simultaneous blocking effect of J30 alone and J30 co-incubated with human VEGGA on the binding of PD-L1 / PD-1 and CD80 / CTLA-4 in a PD-1 / CTLA-4 dual-expression luciferase reporter gene assay.

[0044] Figure 10 The bar chart shows the fold change in IL-2 and IFN-γ secretion by human CD4+ T lymphocytes relative to the blank control.

[0045] Figure 11 The study showed the expression of ICOS and Ki-67 on CD4 and CD8 T cells in a repeated-dose toxicity study of GLP. Detailed Implementation

[0046] The above-described features and advantages of the invention, as well as other features and advantages thereof, will become more clearly understood in conjunction with the accompanying drawings through the following detailed description of embodiments.

[0047] 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. Throughout the specification, identical or similar elements and elements having identical or similar functions are indicated by similar reference numerals.

[0048] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to those skilled in the art. For example, refer to standard manuals such as Leuenberger, HGW, Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2ndEd.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and WileyInterScience, New York (1987); Roitt et al., "Immunology (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background techniques cited above.

[0049] As used herein, unless the context clearly indicates otherwise, the singular forms “a” and “the” also include plural indicators. Thus, for example, references to “antibody” include multiple antibodies.

[0050] Unless otherwise indicated or defined, the term "comprising / including" and its variations should be understood to imply the inclusion of the described element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps. The term "comprising / including" encompasses both "comprising / including" and "consisting". For example, a composition "comprising" X may consist of only X, or may include something else, such as X+Y.

[0051] The term “about” for the numerical value x is optional and means, for example, x+10% or x±5%.

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each of their heavy and light chains. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody can 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 pathway of complement activation). Most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the portion of the antibody that binds to the antigen.

[0053] The light chain variable region (VL) or heavy chain variable region (VH) contains four frame regions, which are separated by three complementarity-determining regions (CDRs). The role of the frame regions is to maintain the spatial conformation of the CDRs, enabling them to specifically bind antigenic epitopes. The CDRs contain the amino acid residues primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following frame (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0054] Assigning amino acids to each VL and VH domain conforms to any conventional definition of CDR. Common 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 & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); complexes of Chothia and Kabat CDRs (also known as Chothia and Kabat combined CDRs), where each CDR is a complex of Chothia and Kabat CDRs; the AbM definition used by the antibody modeling software of Oxford Molecular; 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.

[0055] This disclosure relates to a CDR defined according to any of these numbering systems, although the preferred embodiment relates to a CDR defined by a combination of Chothia and Kabat.

[0056] Table 1. Definition of CDR for antibodies (see http: / / bioinf.org.uk / abs / )

[0057]

[0058] In Table 1, Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system); and Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain, from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system). For example, L24-L34 can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position 24 to position 34 (according to the Chothia numbering system); and H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain, from position 26 to position 32 (according to the Chothia numbering system).

[0059] As used herein, the term "antigen-binding fragment" for antibodies refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0060] Examples of antigen-binding fragments of antibodies 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 connected by a disulfide bridge in the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a portion of the hinge region (see Fundamental Immunology (Paul ed., 3.sup.rd ed. 1993)); (iv) Fd fragments, which consist 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; and (vii) dAb fragments (Ward et al., (1989) Nature). (341:544-546), which consists of a VH domain; (viii) a separate complementarity-determining region (CDR); and (ix) a nanobody, i.e., a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using a recombination approach via a synthetic linker, which allows 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); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" in the term antibody. In addition, the term also includes "linear antibody" which comprises a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form an antigen-binding region, and a modified form of any of the aforementioned fragments that retains antigen-binding activity.

[0061] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for efficacy in the same manner as for intact antibodies.

[0062] The term "valence" in antibody terminology refers to the total number of antigen-binding sites within an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a monovalent antibody contains a total of one antigen-binding site. In an embodiment, the monovalent binding region of a multispecific antibody contains one VH region and one VL region.

[0063] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or cohesion. Affinity, expressed as the equilibrium constant (KD) for the dissociation of the antigen and antibody, is a measure of the strength of binding between an antigenic determinant (epitope) and an antigen-binding site on the antibody: the smaller the KD value, the stronger the binding between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0064] Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity relates to both the affinity between an antigenic determinant (epitope) and its antigen-binding site on the antibody, and the number of associated binding sites present on the antibody. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biolayer interference (BLI) assays, and sandwich competitive assays, as well as various variants thereof known in the art.

[0065] The term "epitope" refers to a site on an antigen that binds to an antibody. Epitopes can be formed from consecutive or non-consecutive 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 upon exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes typically consist of at least three amino acids in a unique spatial conformation, and more typically, at least five or eight to ten amino acids. An epitope defines the minimal binding site of an antibody and is therefore a specific target for the antibody or its antigen-binding fragment.

[0066] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions in an alignment. For example, "an amino acid sequence has X% identity with SEQ ID NO: Y" means that the amino acid sequence has an identity percentage with SEQ ID NO: Y, and states that X% of the residues in the amino acid sequence are identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, computer programs are used for such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0067] In addition, in determining the degree of sequence identity between two amino acid sequences, a technician may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which have little or no effect on the function, activity or other biological properties of the polypeptide.

[0068] Such conservative substitutions are preferably those in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar, or micropolar 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.

[0069] The particularly preferred conservative substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted... Ile or replaced by Val; Lys replaced by Arg, replaced by Gln or replaced by Glu; Met replaced by Leu, replaced by Tyr or replaced by Ile; Phe replaced by Met, replaced by Leu or replaced by Tyr; Ser replaced by Thr; Thr replaced by Ser; Trp replaced by Tyr; Tyr replaced by Trp; and / or Phe replaced by Val, replaced by Ile or replaced by Leu.

[0070] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies. That is, the antibodies constituting the group are identical, except for a small number of possible naturally occurring mutations. Monoclonal antibodies are highly specific and target a single antigen. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology and should not be construed as requiring antibodies to be produced by any particular method.

[0071] In the context of this invention, the term "trispecific antibody" should be understood as an antibody having three different antigen-binding regions defined by different antibody sequences.

[0072] As used in this article, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and is therefore used to target cancer cells.

[0073] As used in this article, the term "carrier" is intended to refer to a nucleic acid molecule that can transport another nucleic acid to which it is attached.

[0074] As used in this article, the term "host cell" refers to a cell in which an expression vector has been introduced.

[0075] The term “pharmaceutical acceptable” means that the carrier or adjuvant is compatible with the other components of the composition and is substantially harmless to the recipient and / or that such carrier or adjuvant is approved or approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0076] As used herein, the term "treatment," etc., refers to the administration of a drug or the performance of an action to achieve a certain effect. The effect may be preventative, i.e., complete or partial prevention of a disease or its symptoms, and / or therapeutic, i.e., partial or complete cure of a disease or its symptoms. In this context, "treatment" may include the treatment of a disease or condition (e.g., cancer) in mammals (particularly humans), encompassing: (a) preventing a subject who may be susceptible to a disease but has not yet been diagnosed with it from developing the disease or its symptoms (e.g., including diseases that may be related to or caused by a primary disease); (b) suppressing the disease, i.e., halting its progression; and (c) alleviating the disease, even if the disease subsides. Treatment may refer to any indication of success in treating, improving, or preventing cancer, including any objective or subjective parameter such as: symptom reduction; disease remission; symptom relief or making the patient's condition more tolerable; a slowing of the rate of degeneration or decline; or making the final stage of degeneration 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, compositions, or conjugates disclosed herein to prevent or delay, alleviate, or prevent or inhibit the occurrence or development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect / efficacy" refers to the reduction, elimination, or prevention of a subject's disease, symptoms of the disease, or side effects of the disease.

[0077] As used in this article, the term "effective amount" means the amount that is sufficient to treat a disease when administered to a subject for the purpose of treating that disease.

[0078] As used herein, the term “subject” means any mammalian subject for whom a diagnosis, treatment, or therapy is desired. “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, domesticated 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.

[0079] Trispecific antibodies

[0080] In one aspect, this disclosure provides a trispecific antibody comprising a first antigen-binding region that binds to PD1, a second antigen-binding region that binds to CTLA4, and a third antigen-binding region that binds to VEGFa, wherein the first antigen-binding region comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), and wherein: the first VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 7, 8, and 9, respectively, and the first VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 10, 11, and 12, respectively.

[0081] In some implementations, the CDR sequence is defined according to the Kabat numbering system.

[0082] When the CDR sequence is defined according to the Kabat numbering system, the first VH 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: 13, and the first VL 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: 14. In a preferred embodiment, the first VH contains the amino acid sequence of SEQ ID NO: 13, and the first VL contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the first antigen-binding region is in the form of an scFv. In some embodiments, the second antigen-binding region comprises a second VH and a second VL. In some embodiments, the second antigen-binding region is in the form of an scFv.

[0083] In some embodiments, the third antigen-binding region comprises a third VH and a third VL. In some embodiments, the third antigen-binding region is in Fab form.

[0084] In some implementations, the first antigen-binding region and the second antigen-binding region are monovalent. In some implementations, the third antigen-binding region is bivalent.

[0085] In some implementations, the trispecific antibody comprises two heavy chains and two light chains, wherein the two heavy chains comprise heavy chain 1 and heavy chain 2, heavy chain 1 comprises a first antigen-binding region in the form of scFv at its C-terminus, and heavy chain 2 comprises a second antigen-binding region in the form of scFv at its C-terminus.

[0086] In some implementations, each of the two heavy chains contains a third antigen-binding region (VH) at its N-terminus, and each of the two light chains contains a third antigen-binding region (VL) at its N-terminus.

[0087] In some embodiments, the trispecific antibody further includes a CH3 constant region. In some preferred embodiments, the trispecific antibody further includes an Fc region.

[0088] In some implementations, the Fc region has an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

[0089] In some implementations, the Fc region has a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0090] In a preferred embodiment, the Fc region is a subtype of IgG1.

[0091] One of the challenges in effectively generating bispecific and trispecific antibody formulations is the mismatch between heavy and light chains when co-expressing chains with different binding specificities. Table 2 lists several amino acid substitutions used to overcome mismatches between heavy chains with different binding specificities, which "enhance" or preferentially promote proper association between the desired heavy chains. The trispecific antibodies of this disclosure can be prepared using any method that prevents or reduces mismatches between heavy chains.

[0092] Table 2

[0093]

[0094] The "mortar and pestle" (KiH) method relies on the modification of the interface between two CH3 domains (where most interactions occur). Typically, a large amino acid residue is introduced into the CH3 domain of one antibody heavy chain, acting like a pestle. On the other heavy chain, a mortar is formed to accommodate this large residue, acting like a keyhole. The resulting heterodimer's Fc moiety can be further stabilized by artificially introduced disulfide bonds.

[0095] The alternative strategy is based on ionic interactions or spatial complementarity between charged residues. This method alters the charge polarity at the CH3 interface, allowing electrostatically matched Fc domains to promote heterodimer formation during co-expression through favorable mutual attraction while retaining the hydrophobic core structure; conversely, unfavorable charge repulsion inhibits homodimerization. See Table 2. The amino acid numbers in Table 2 follow the Kabat numbering rules and are applicable to the heavy chain amino acid sequences of the antibodies described herein.

[0096] In addition, leucine zipper (LZ) domains can be introduced into protein scaffolds. Leucine zippers are a common three-dimensional structural motif in proteins, often serving as part of the DNA-binding domain of various transcription factors. A single leucine zipper typically contains 4-5 leucine residues, spaced approximately 7 amino acids apart, forming an amphiphilic α-helix with its hydrophobic region distributed along one side of the helix. In one specific embodiment, the heterodimeric protein scaffold comprises a leucine zipper from the c-jun transcription factor combined with a leucine zipper from the c-fos transcription factor. Although c-jun is known to form jun-jun homodimers while c-fos cannot, the formation of jun-fos heterodimers is far more advantageous than that of jun-jun homodimers.

[0097] The leucine zipper domain can be incorporated in place of the CH2-CH3 sequence in the protein scaffold, or it can be positioned at the C-terminus of the two heavy chains in a trispecific antibody. In the latter case, a furin cleavage site can be introduced between the C-terminus of CH3 and the N-terminus of the leucine zipper. This can facilitate furin-mediated leucine zipper cleavage following the heterodimerization step when the heavy and light chains of the trispecific antibody are co-expressed in a suitable mammalian cell expression system (see Wranik et al., J. Biol. Chem., 287(5):43331-43339, 2012).

[0098] In some implementations, the CH3 constant region contains a club-and-mortar mutation.

[0099] In some embodiments, the second antigen-binding region comprises a second VH and a second VL, wherein the second VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 15, 16 and 17, respectively, and the second VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 18, 19 and 20, respectively.

[0100] In some embodiments, the second VH 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: 21, and the second VL 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: 22. In a preferred embodiment, the second VH comprises the amino acid sequence of SEQ ID NO: 21, and the second VL comprises the amino acid sequence of SEQ ID NO: 22.

[0101] In some embodiments, the third antigen-binding region comprises a third VH and a third VL, wherein the third VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 23, 24 and 25, respectively, and the third VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 26, 27 and 28, respectively.

[0102] In some embodiments, the third VH 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: 29, and the third VL 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: 30. In a preferred embodiment, the third VH comprises the amino acid sequence of SEQ ID NO: 29, and the third VL comprises the amino acid sequence of SEQ ID NO: 30.

[0103] In some embodiments, heavy chain 1 includes, from the N-terminus to the C-terminus: a third antigen-binding region (VH, CH1, Fc regions) and a first antigen-binding region in the form of scFv; heavy chain 2 includes, from the N-terminus to the C-terminus: a third antigen-binding region (VH, CH1, Fc regions) and a second antigen-binding region in the form of scFv; and each of the two light chains includes, from the N-terminus to the C-terminus: a third binding region (VL) and a light chain constant region.

[0104] Based on the amino acid sequence of the constant region of the light chain, the light chain of an antibody can be classified as a lambda (λ) chain or a kappa (κ) chain.

[0105] In some embodiments, heavy chain 1 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: 32; heavy chain 2 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: 33; and each of the two light chains 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: 31.

[0106] In a preferred embodiment, the trispecific antibody comprises two heavy chains and two light chains, wherein heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 32; heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 33; and each of the two light chains comprises the amino acid sequence of SEQ ID NO: 31.

[0107] Nucleic acid

[0108] This disclosure provides a nucleic acid encoding the trispecific antibody disclosed herein.

[0109] The term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or any type of modified form of nucleotides. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linking modifications such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, thioaniline phosphates, aniline phosphates, and phosphoroamidates.

[0110] This invention provides nucleic acid molecules encoding the heavy chain sequences disclosed herein. For example, this invention provides nucleic acid molecules encoding sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the heavy chain sequences disclosed herein. In some embodiments, the nucleic acid encodes a 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: 32.

[0111] This invention provides nucleic acid molecules encoding the heavy chain sequences disclosed herein. For example, this invention provides nucleic acid molecules encoding sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the heavy chain sequences disclosed herein. In some embodiments, the nucleic acid encodes a 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: 33.

[0112] This invention provides nucleic acid molecules encoding the light chain sequences disclosed herein. For example, this invention provides nucleic acid molecules encoding sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the light chain sequences disclosed herein. In some embodiments, the nucleic acid encodes a 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: 32.

[0113] The present invention also provides nucleic acid molecules encoding the heavy chain variable region sequences disclosed herein. For example, the present invention provides nucleic acid molecules encoding sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the heavy chain variable region sequences disclosed herein.

[0114] In some embodiments, the nucleic acid encoding a 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: 13. In some embodiments, the nucleic acid encoding a 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: 21. In some embodiments, the nucleic acid encoding a 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: 29.

[0115] The present invention also provides nucleic acid molecules encoding the light chain variable region sequences disclosed herein. For example, the present invention provides nucleic acid molecules encoding sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the light chain variable region sequences disclosed herein.

[0116] In some embodiments, the nucleic acid encoding a 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: 14. In some embodiments, the nucleic acid encoding a 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: 22. In some embodiments, the nucleic acid encoding a 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: 30.

[0117] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.

[0118] 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 vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

[0119] In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0120] carrier

[0121] This disclosure provides vectors containing the nucleic acids disclosed herein.

[0122] In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy chain variable region or a light chain variable region of an antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

[0123] 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.1 GFP, 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.

[0124] Expression vectors 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, LaJolla, 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 useful in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0125] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Constructs of circular or linear expression vectors can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

[0126] For example, the vector could be an adenoviral vector containing a nucleotide sequence encoding an antibody disclosed herein. The vector could be administered to a subject and then enter the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the cell's genome, and subsequently the cells expressing the antibody disclosed herein.

[0127] host cells

[0128] This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.

[0129] Any cell can be used as a host cell for the nucleic acids or vectors used in this disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a 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, for example, Enterobacteriaceae such as Escherichia, e.g., Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; and Pseudomonas such as Pseudomonas aeruginosa. (Aeruginosa); and Streptomyces. In some implementations, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g., 293 or 293 cells for subclones grown in suspension culture), Expi293™ cells, CHO cells, juvenile hamster kidney cells (e.g., BHK, ATCC CCL10), mouse testicular supporting cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL70), African green monkey kidney cells (e.g., VERO-76, ATCCCRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL2), canine kidney cells (e.g., MDCK, ATCC CCL34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CCL1442), human lung cells (e.g., W138, ATCCCCL75), human hepatocytes (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCCCCL51), TRI cells, MRC cells, etc. 5 cells, FS4 cells, human hepatocellular carcinoma lines (e.g., Hep G2) and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0130] The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the antibodies disclosed herein in vivo.

[0131] This invention provides a host cell introduced therein from any of the above-described vectors. The invention further provides a method for preparing the antibody of the invention, wherein the method comprises a) culturing the host cell disclosed herein under conditions suitable for antibody production; and b) obtaining the antibody from the culture.

[0132] Composition

[0133] This disclosure provides pharmaceutical compositions comprising the trispecific antibody disclosed herein, and optionally at least one pharmaceutically acceptable carrier or excipient.

[0134] The antibodies or pharmaceutical agents of the present invention (also referred to herein as “active compounds”) and their derivatives, fragments, analogs, and homologues may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an antibody or pharmaceutical agent and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, buffers, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer solutions.

[0135] In some embodiments, the composition further comprises a second therapeutic agent, optionally selected from the group consisting of free antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, peptides, and small molecule drugs.

[0136] Medical use

[0137] On the other hand, this disclosure provides methods for treating a disease, comprising administering to a subject an effective amount of the disclosed trispecific antibody or the disclosed pharmaceutical composition. In some embodiments, the disease is cancer.

[0138] This disclosure provides the use of the trispecific antibodies or pharmaceutical compositions disclosed herein in the preparation of medicaments for treating a disease. In some embodiments, the disease is cancer.

[0139] This disclosure also provides the trispecific antibodies disclosed herein, or the pharmaceutical compositions disclosed herein, for the treatment of diseases. In some embodiments, the disease is cancer.

[0140] The therapeutically effective amount of the composition is administered to the subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to suppress the signs or symptoms of cancer.

[0141] In some embodiments, the cancer is CTLA4-associated cancer. In some embodiments, the cancer is VEGFa-associated cancer. In some embodiments, the cancer is PD-L1-expressing cancer. Suitable subjects may include those diagnosed with PD-L1-expressing cancer. In some embodiments, the cancer is PD-L1 overexpressing. In some embodiments, the disease is PD-L1 negative. In some preferred embodiments, the cancer is a solid tumor.

[0142] In some implementations, solid tumors include, but are not limited to, colon cancer, adenocarcinoma of the rectum and colon, prostate cancer, breast cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), liver cancer (including hepatocellular carcinoma), stomach cancer, renal cancer, kidney cancer (including renal cell carcinoma), cervical cancer, ovarian cancer, and endometrial cancer.

[0143] The precise therapeutically effective dose for human subjects will depend on the severity of the disease state, the subject's general health, age, weight and sex, diet, timing and frequency of administration, drug combination, sensitivity to response, and tolerance / response to the therapy. This dose can be determined through routine laboratory testing and is within the judgment of the clinician. Typically, the therapeutically effective dose can be from 0.01 mg / kg to 50 mg / kg, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 mg / kg. Pharmaceutical compositions can be conveniently presented in unit doses containing a predetermined amount of the active agent of the invention.

[0144] The dosage of the antibody molecules disclosed herein depends on the nature of the condition to be treated, the degree of inflammation present, and whether the antibody molecules are being used prophylactically or to treat an existing condition.

[0145] The frequency of administration will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (e.g., 2 to 10 hours), daily administration of one or more doses may be necessary. Alternatively, if the antibody molecule has a long half-life (e.g., 2 to 15 days), once-daily, once-weekly, or even once every one or two months may be necessary. In some embodiments, the trispecific antibody is administered once weekly. In some embodiments, the trispecific antibody is administered once every two weeks. In some embodiments, the trispecific antibody is administered once every three weeks.

[0146] Example

[0147] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention. Experimental procedures not specified in the following embodiments are performed according to conventional procedures and conditions, or according to the specification.

[0148] Example 1: Construction, expression, and purification of anti-PD1 / CTLA-4 / VEGF trispecific antibody J30

[0149] 1-1 J30 sequence and structure

[0150] This invention constructs a trispecific antibody J30 capable of recognizing PD1, CTLA-4, and VEGFA, derived from anti-PD1 antibody CS1003, anti-CTLA-4 antibody CS1002, and anti-VEGFA antibody I10. The structure of the constructed J30 is as follows. Figure 1 As shown in Table 12, the constant region sequence of human hIgG1 was used to construct the trispecific antibody of this invention.

[0151] 1-2 Expression and Generation

[0152] The corresponding polynucleotide sequence of the trispecific antibody J30 was synthesized and inserted into the expression vector pCDNA3.4. This vector was then expressed as human IgG1 in Chinese hamster ovary (CHO) cells and subsequently purified using protein A magnetic beads according to the manufacturer's instructions. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm. The control antibodies used in this example were constructed as follows and expressed and purified according to the methods described above. The following antibodies were used as positive controls in this example: AK112 (Ivonescimab, PD1 / VEGF bispecific antibody), AK104 (Cadonilimab, PD1 / CTLA4 bispecific antibody), MEDI5752 (Volrustomig, PD1 / CTLA4 bispecific antibody), pembrolizumab (anti-PD1 antibody), and bevacizumab (anti-VEGF antibody).

[0153] Example 2: Detection of binding affinity of J30 to PD1, CTLA4 and VEGFA proteins using Octet.

[0154] J30 and isotype human IgG1 were measured using a ForteBio Octet RED96e and HIS1K biosensor (Sartorius #18-5120) via biolayer interferometry (BLI). The assay was performed at 30°C with mixing at 1000 rpm. All samples were diluted in 1x kinetic buffer prepared with 10x kinetic buffer (Sartorius #18-1105) and PBS. The sensor was loaded with 5 μg / mL PD1 protein (SinoBiological #10377-H08H) or CTLA4 protein (SinoBiological #11159-H08H) for 240 seconds, followed by baseline equilibration with 1x kinetic buffer for 180 seconds. Association between J30 and isotype Abs was performed at 200 nM, 20 nM, 2 nM, and 0 nM for 300 seconds, followed by dissociation with 1x kinetic buffer for 300 seconds. Analyze the data using ForteBio data analysis software HT12.0.

[0155] J30 and its isotype were also tested via BLI using a ForteBio Octet RED96e and an anti-hIgG Fc Capture (AHC) biosensor (Sartorius #18-5060). The assay was performed at 30°C with mixing at 1000 rpm. All samples were diluted in 1x kinetic buffer prepared with 10x kinetic buffer (Sartorius #18-1105) and PBS. The sensor was loaded with 10 μg / mL J30 or the isotype Ab for 120 seconds, followed by baseline equilibration with 1x kinetic buffer for 120 seconds. Association with recombinant VEGF121 protein (SinoBiological #10008-HNAH) was performed at 100 nM, 33 nM, 11 nM, and 0 nM for 120 seconds, followed by dissociation with 1x kinetic buffer for 300 seconds. Data were analyzed using ForteBio data analysis software HT12.0.

[0156] The OCTET binding results are shown in Table 3. J30 exhibits double-digit nanomolar affinities for PD1 and CTLA4.

[0157] Table 3. Summary of binding affinities of J30 to PD1, CTLA4, and VEGFA

[0158]

[0159] Example 3: Detection of multi-target binding of J30 to PD1, CTLA4 and VEGFA proteins using Octet

[0160] Simultaneous triple binding with PD1, CTLA4, and VEGFA was measured via BLI. J30, I10, and isotype human IgG1 were measured using a ForteBio OctetRED96e and an SA biosensor (Sartorius #18-5019). The assay was performed at 30°C with mixing at 1000 rpm. All samples were diluted in 1x kinetic buffer prepared with 10x kinetic buffer (Sartorius #18-1105) and PBS. The sensor was loaded with 5 μg / mL VEGF165 protein (SinoBiological #11066-H27H-B) for 120 seconds, followed by baseline equilibration with 1x kinetic buffer for 120 seconds. Association of J30, I10, and isotype human IgG1 was performed at 200 nM for 240 seconds, followed by dissociation with 1x kinetic buffer for 30 seconds. Then, association was initiated with 5 μg / mL PD1-mFc protein (ACRO #PD1-H5255) for 240 seconds, followed by dissociation with 1x kinetic buffer for 30 seconds. Finally, association was initiated with 5 μg / mL CTLA4 protein (SinoBiological #11159-H08H) for 240 seconds, followed by dissociation with 1x kinetic buffer for 30 seconds. Data were analyzed using ForteBio data analysis software HT12.0. OCTET binding results showed... Figure 2 In the middle, J30 showed simultaneous binding with PD1, CTLA4, and VEGFA.

[0161] Example 4: Detection of the binding and blocking activity of J30 on PD1, CTLA4 single-positive and double-positive cells by FACS

[0162] The following cell lines were used: PD1 single-positive cells NK92-PD1-GFP (Celetrix), CTLA4 single-positive cells CHO-K1-CTLA4 (Kyinno Bio), and PD1 / CTLA4 double-positive cells CHO-S-PD1-CTLA4 (Sanyou Bio). A CHO-S-PD1-CTLA4 cell line with a PD1 / CTLA4 ratio of approximately 40:1 was selected, a ratio similar to that found on tumor-infiltrating lymphocytes (TILs). These three cell lines were used to assess the binding and ligand blocking activity of J30 compared to the CS1003 and CS1002 combination.

[0163] For CHO-K1-CTLA4 and CHO-S-PD1-CTLA4, RPMI-1640 medium containing 10% fetal bovine serum was used; for NK92-PD1-GFP, NK92 medium was used. All cells were cultured at 37°C with 5% CO2. Cells were harvested during the logarithmic growth phase. Cell density was assessed using CounterStar and adjusted to 2 x 10⁶ cells / cells with culture medium. 6 Cells / mL. Pack 2 x 10 cells / mL in each well. 5 Cells were seeded into 96-well U-plates. Cells were washed twice with staining buffer (BD #554656) and precipitated at 300 xg and 2–8°C for 3 minutes. Cells were resuspended and incubated for 30 minutes at 2–8°C in the dark with 100 μL / well of cold-diluted antibody in staining buffer as the first antibody solution. The antibody concentration was tested starting at 200 nM and serially diluted 7 times in 5-fold increments. After 30 minutes of incubation at 2–8°C, cells were washed and fixed at 37°C for 10 minutes with fresh Phosflow™ lysis / fixation buffer (1x) (BD #558049) pre-warmed to 37°C. After washing twice, cells were resuspended and incubated at 2–8°C in the dark for 30 minutes with 100 μL / well of cold ligand (either 100 nM human PD-L1-mFc (Acro #PD1-H52A3) or 100 nM human B7-1-mFc (Acro #B71-H52A4)). Unbound PD1 receptors were detected using the PD1 ligand PD-L1-mFc, while unbound CTLA4 receptors were detected using the CTLA4 ligand B7-1-mFc. After washing twice, cells were resuspended and incubated with a mixture of 100 μL / well of goat anti-mouse IgG AF647 (1:1000) (Jackson #115-605-062) and anti-human IgG PE secondary antibody (1:100) (Invitrogen #12-4998-82) as a secondary antibody, and incubated at 2–8°C in the dark for 30 minutes. After washing twice with staining buffer, cells were resuspended in 100 μl / well of staining buffer and analyzed using a Beckman CytoFlex flow cytometer. Nonlinear regression analysis with four adjustable parameters was performed using GraphPad Prism 9.2 software.

[0164] The relevant results show Figure 3Figures ab show the results for PD1 / CTLA4 double-positive CHO-S-PD1-CTLA4 cells; Figures cd show the results for CTLA4 single-positive CHO-K1-CTLA4 cells; and Figure ef shows the results for PD1 single-positive NK92-PD1-GFP cells. On CHO-S-PD1-CTLA4 cells, J30 blocked B7 / CTLA4 interaction with greater potency than the combination group. Simultaneously, the monovalent anti-PD1 arm of J30 remained functional. The monovalent anti-CTLA4 arm of J30 did not block B7 / CTLA4 interaction on CHO-hCTLA4 cells. J30 will be able to re-invigorate PD1 / CTLA4 double-positive tumor-infiltrating T cells without over-activating peripheral CTLA4 single-positive T cells, thereby avoiding immune-related adverse events (irAEs).

[0165] Example 5: Detection of the blocking effect of J30 on the binding of VEGFA and VEGFR2 by the VEGFR2-NFAT reporter gene.

[0166] The HEK293 / VEGFR2 / NFAT-luciferase cell line (Genscript #M00891) was used to detect the blocking ability of J30 on the binding of human VEGFA and human VEGFR2. The HEK293 / VEGFR2 / NFAT-luciferase cell line is a luciferase reporter gene cell line constructed based on the NFAT signaling pathway. When VEGFA binds to VEGFR2, it triggers the NFAT signaling pathway, thereby inducing luciferase expression.

[0167] For HEK293 / VEGFR2 / NFAT-luciferase reporter cells, DMEM medium containing 10% FBS, 1 μg / ml puromycin, and 200 μg / ml hygromycin B was used. HEK293 / VEGFR2 / NFAT-luciferase cells were digested with acutase and resuspended in basal medium (DMEM + 1% heat-inactivated FBS). Cell density was assessed using CounterStar and adjusted to 1x10⁻⁶ cells / cells. 6 Cells / well, and 40 μL of cell culture is seeded into 96-well plates, which is 4 x 10 cells per well. 4Cells were tested. 80 μL of 40 ng / mL hVEGF165-His (Genscript # Z03073-10) was mixed with 80 μL of antibody solution and incubated at room temperature for 30 min. Antibody concentrations were tested starting at 400 nM and serially diluted 4-fold eight times. 40 μL of the mixture was added to each well of a 96-well plate and incubated at 37°C for 6 h. Therefore, the final concentration of hVEGF165-his was 10 ng / mL, and the final starting concentration of each antibody was 100 nM. After incubation, 80 μL / well luciferase buffer (Beyotime #RG051M) was added, and the luminescence signal was detected under the following conditions: gain (200) and measurement interval (1 s).

[0168] The results show Figure 4 As shown in Table 4, J30 blocks the interaction between human VEGFA and human VEGFR2 on the cell surface (with effects comparable to I10 and AK112), thereby inhibiting downstream VEGFR2 signaling.

[0169] Table 4 Summary of blocking activities in HEK293 / VEGFR2 / NFAT-luciferase cell lines

[0170]

[0171] Example 6: Function of J30 in inhibiting VEGFA-driven proliferation of human umbilical vein endothelial cells (HUVECs)

[0172] Human umbilical vein endothelial cells (HUVECs) express VEGF receptors and are therefore VEGF-induced to proliferate, making them useful for evaluating the angiogenic effects of anti-VEGF antibodies. HUVECs (ATCC, CRL-1730) were used to detect the inhibition of VEGF function by a trispecific antibody.

[0173] HUVEC cells were seeded into 96-well clear plates and adjusted to 3x10⁻⁶ cells / well. 3 Cells / well. Antibody concentrations were started at 10 nM and serially diluted 8 times using a 2-fold dilution factor. The serially diluted antibody was mixed with VEGFA (Sino Biological, #11066-HNAH-100ug) to a final concentration of 20 ng / mL and incubated at room temperature for 30 min. 50 μL of the mixture was added to each well of a 96-well plate and incubated at 37°C for 72 h. After 72 h of incubation, 20 μL / well MTS was added to the plate and incubated at 37°C for 4 h. Fluorescence signals were read at OD492. The IC50 of the antibody inhibitory activity was calculated using a four-parameter model. 50 Value. The result is displayed. Figure 5 As shown in Table 5, J30 effectively blocked VEGFA-driven HUVEC cell proliferation, with IC50 showing a significant effect. 50 It is 1.012 nM.

[0174] Table 5. Summary of inhibitory activities on HUVEC cell growth

[0175]

[0176] Example 7: J30 internalization of PD1 and CTLA4

[0177] To evaluate the internalization rate of PD1 and CTLA4 on the cell surface, CHO-S-PD1-CTLA4 cells (Sanyou Bio) were used to test J30, AK104, AK112, MEDI5752, and allotype human IgG1.

[0178] CHO-S-PD1-CTLA4 cells were tested with 200 nM antibody solution at 0 min, 15 min, 30 min, and 60 min using an indirect internalization method (FACS surface binding). For the 0 min internalization group (which served as the initial value), the FACS procedure was the same as described in Example 4. For the 15 min, 30 min, and 60 min internalization groups, after incubation with the first antibody solution, cells were washed twice with 200 μL / well of cold medium, resuspended with 100 μL / well of medium, and cultured at 37°C at the specified time points. After washing twice with staining buffer, the collected cells were stained with the second antibody. The subsequent FACS procedure was similar to that in Example 4. The internalization rate at 15 min, 30 min, and 60 min was calculated as 100% - (MFI at the specified time point / MFI at 0 min)%.

[0179] The results of the indirect method are shown Figure 6 In this study, all tested antibodies were internalized on CHO-S-PD1-CTLA4 cells. Target internalization reduces the number of available targets on the cell surface, thereby reducing inhibitory signaling. J30 demonstrated superior performance compared to other tested antibodies.

[0180] Example 8: In vivo efficacy of J30 in inhibiting MC-38-PDL1 growth in humanized PD1 / PDL1 / CTLA4 mice

[0181] For the in vivo efficacy evaluation of J30, 5x10 5One B-hPD-L1 MC38 plus cell (Biocytogen) was subcutaneously injected into female B-hPD1 / hPD-L1 / hCTLA4 humanized (C57BL / 6) mice (Biocytogen). Mice were randomly assigned to study groups, and once the tumor reached approximately 99 mm... 3 The test sample is administered via intraperitoneal injection. Tumor volume is calculated using the following formula: Volume = ½ x Length x Width x Width. Tumor growth inhibition (TGI) of tumor volume is calculated using the following equation: TGI% = [1 - (T...] i -T0) / (C i -C0)]x100%. (T i T0: Mean tumor volume of the treatment group on day i after treatment; C i C1: Mean tumor volume of the control group on day i post-treatment; C2: Mean tumor volume of the control group on day 0. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the animal facility. Efficacy study grouping information is listed in Table 6.

[0182] On day 29 post-treatment, 2.4 mg / kg J30 induced a 60% TGI, compared to 33.3%, 28.2%, and 8.4% TGI for equimolar doses of MEDI5752, AK112, and AK104, respectively. Combination groups (pembrolizumab / CS1002 Fc null and CS1003 / CS1002 Fc null) resulted in 37.5% and 27.6% TGI, respectively. No weight loss or treatment-related toxicities were observed during the study.

[0183] Typically, J30 exhibits superior tumor growth inhibition in B-hPD1 / hPD-L1 / hCTLA4 humanized mice compared to MEDI5752, AK112, AK104, pembrolizumab / CS1002 combination, and CS1003 / CS1002 combination.

[0184] Table 6. List of study groups investigating the efficacy of MC-38-PDL1 in humanized PD1 / PDL1 / CTLA4 transgenic mouse models

[0185]

[0186] Example 9: In vivo efficacy of J30 in inhibiting the growth of MC38-hVEGFA in humanized CTLA4 / VEGFR2 mice

[0187] J30 exhibits binding activity against mouse PD1. The in vivo antitumor efficacy of J30 was also evaluated in a subcutaneous B-hVEGFA MC38 (V2) mouse colon cancer model (human VEGFA-transduced MC38 cells) from the B-hCTLA4 / VEGFR2 transgenic (C57BL / 6) mouse model (Biocytogen). Mice were randomly assigned to study groups, and treatment was initiated once the tumor reached approximately 99 mm. 3 The test sample was administered via intraperitoneal injection. Tumor volume was calculated using the formula (volume = ½ x length x width x width). No weight loss or treatment-related toxicities were observed during the study. All animal procedures were performed according to protocols approved by the animal facility's Institutional Animal Care and Use Committee. Grouping information is listed in Table 7.

[0188] On day 28 post-treatment, the median tumor volume in the 3 mg / kg J30 group was 527 mm. 3 In the equimolar dose group of AK112 and the combination group of CS1002 Fc null, CS1003 and bevacizumab, the median tumor volume was 730 mm. 3 and 640 mm 3 In the 12 mg / kg or equimolar dose groups, the median tumor volume in the J30, AK112, and CS1002 Fc null, CS1003, and bevacizumab combination groups was 354 mm. 3 565 mm 3 and 459 mm 3 Compared to the control group, J30 showed significant inhibition of tumor growth in a dose-dependent manner at the tested dose. Compared to other groups administered with equimolar amounts of the drug, J30 exhibited superior antitumor effects compared to the combination groups of AK112 and CS1002, CS1003, and bevacizumab. Figure 8 a and 8b).

[0189] On day 35 (11 days after the last dose), all treated animals survived except for one animal from the combination groups of 2.25 mg / kg CS1002 Fc null, 2.25 mg / kg CS1003, and 2.25 mg / kg bevacizumab. J30 showed an effect on the survival of tumor-bearing mice. Kaplan-Meier survival curves for all groups are shown at 8c and 8d.

[0190] In summary, J30 demonstrated a significant, dose-dependent inhibitory effect on the growth of subcutaneous B-hVEGFA MC38(V2) mouse colon cancer at the tested dose. Compared to other treatment groups administered with equimolar amounts of the drug, the antitumor effect of J30 was comparable to that of the combination groups of AK112 and CS1002 Fc null, CS1003, and bevacizumab, showing a slight trend towards superiority. Furthermore, all tested products exhibited potent antitumor efficacy with no significant adverse reactions, indicating good tolerability.

[0191] Table 7. List of efficacy study groups for MC38-hVEGFA in B-hCTLA4 / VEGFR2 transgenic mouse models.

[0192]

[0193] Example 10: Enhanced crosslinking between J30 and VEGFA dimers, assessed by a PD-1 / CTLA-4 dual reporter gene assay, blocks PD-1 and CTLA-4 mediated checkpoint signaling.

[0194] VEGFA is a secreted functional dimer in which two monomers are linked together by disulfide bridges between two subunits. To determine whether VEGFA could enhance the blocking activity of J30, we evaluated the ability of J30 alone and J30 co-incubated with human VEGFA dimer (J30+VEGFA) to block the binding of PD-L1 / PD-1 and CD80 / CTLA-4. The blocking effect of J30 or J30+VEGFA on the interaction between PD-L1 / PD-1 and CD80 / CTLA-4 was detected using H_CTLA4 PD-1 reporter cells (Genomeditech # HC142) and H_CD80 PDL1 aAPCCHO-K1 cells (Genomeditech # MC080) as antigen-presenting cells (APCs). The H_CD80 PDL1 aAPC CHO-K1 cell line is a constitutively stable clonal cell line expressing OKT3, human PDL1, and human CD80 genes. This cell line was compared with H_CTLA4... PD-1 reporter cell line (GM-C26486) was co-cultured. OKT3 binding to TCR and CD80 binding to CD28 jointly activate T cell signaling. PD-L1 binds to PD-1, or CTLA-4 competes with CD28 for CD80, thereby blocking luciferase expression. Blocking antibodies can block this inhibitory signaling and restore T cell activation. Luciferase activity measurements indicate the activation level of signaling pathways and can therefore be used to evaluate the in vitro effects of drugs associated with CTLA4 and PD1.

[0195] H_CD80 PDL1 aAPC CHO-K1 cells were resuspended in culture medium (containing 10% FBS, 1% PS, 4 μg / mL puromycin, 4 μg / mL blastomycin, and 100 μg / mL hygromycin B in F12K) to a concentration of 1.5 x 10⁻⁶. 5 Cells / mL were collected, and 100 μL of cells were added to each well of a 96-well microplate (Corning 3903). After overnight incubation at 37°C with 5% CO2, the culture medium was discarded from the 96-well microplate, and 50 μL of serially diluted antibody sample was added to each well. For J30+VEGFA preparation, J30 was mixed with human VEGF165 protein and incubated at room temperature for 30 minutes. Then, 50 μL of 10... 5 H_CTLA4 PD-1 reporter cells were added to each well. Additionally, 96-well plates containing both cell lines were incubated at 37°C and 5% CO2 for 7 h. After incubation, 100 μL / well of luciferase substrate was added to all wells. LUM (luminescence) signals were read using a microplate reader (BioTek, Synergy 2) at a gain of 200 and a measurement interval of 1 second. A scatter plot of LUM was plotted using GraphPad 10.3.1 software, and EC50 was calculated. 50 .

[0196] Both J30 and J30+VEGFA can simultaneously block the binding of PD-L1 / PD-1 and CD80 / CTLA-4, thereby releasing TCR signaling activity to trigger luciferase expression, with EC50 values ​​of 73.96 nM and 4.41 nM, respectively. 50 (Table 8, Figure 9 In summary, through cross-linking with VEGFA dimers, J30 achieved a 16.8-fold increase in its function as an immune checkpoint inhibitor (CPI).

[0197] Table 8. EC5 values ​​of J30 and J30+VEGFA alone in PD-1 / CTLA-4 dual-expression luciferase reporter gene assays 50 value

[0198]

[0199] Example 11: Evaluation by MLR assay of the enhanced crosslinking between J30 and VEGFA dimers on the blocking of PD-1 / PD-L1 and CTLA4 / CD80 signaling.

[0200] Allogeneic mixed lymphocyte response (MLR) assay was used to evaluate the effect of J30 alone or J30+VEGFA on CD4. +T lymphocyte activation. The density of human isolated CD4+ T cells and myeloid dendritic cells (MDCs) was adjusted to 1.2 x 10⁻⁶ using RPMI-1640 containing 10% FBS. 6 cells / mL and 4 x 10 5 Cells / mL. Then, 100 μL / well of CD4 + T cells were added to 96-well plates, and then 50 μL of serially diluted antibody sample was added to each well. For J30+VEGFA, the antibody was mixed with human VEGF165 protein and incubated at room temperature for 30 min. Then 100 μL / well of mDC was added to each well to produce a final volume of 250 μL per well. After incubation at 37°C and 5% CO2 for 3 days, 120 μL of supernatant was collected for IL-2 detection. After incubation at 37°C and 5% CO2 for another 2 days, 80 μL of supernatant was collected for IFN-γ detection. The fold change in IL-2 and IFN-γ secretion levels in the supernatant samples relative to the blank control was calculated and plotted as a bar chart using GraphPad 10.3.1 software.

[0201] In allogeneic MLR, J30 alone and J30+VEGFA showed a dose-dependent promotion of CD4. + The potential for T lymphocyte proliferation and activation. At all tested concentrations, J30+VEGFA showed a stronger ability to enhance IL-2 secretion compared to J30 alone. Figure 10 a). For IFN-γ secretion, J30+VEGFA showed a stronger promoting effect than J30 at 1 nM ( Figure 10 b). These results indicate that co-incubation of J30 with human VEGFA dimer enhances J30's ability to activate T lymphocytes.

[0202] Example 12: Single-dose pharmacokinetics (PK) and repeated-dose toxicokinetics (TK) of J30 in cynomolgus monkeys

[0203] Based on in vitro cross-species binding affinity data, cynomolgus monkeys were identified as a pharmacologically relevant species. Therefore, a non-clinical PK evaluation of J30 was performed in cynomolgus monkeys. IV infusion, the anticipated clinical route of administration, was used as the route of administration in this embodiment. The complete PK was evaluated in independently run single-dose studies with clinically relevant dose ranges. Multiple-dose cynomolgus monkey toxicokinetics (TK) studies and repeated-dose toxicity studies were conducted concurrently.

[0204] The pharmacokinetic (PK) characteristics of J30 were investigated following a single IV infusion of 5, 15, or 50 mg / kg (n=6 / dose level). Serum concentrations were monitored for 4 weeks, and parameters were estimated by non-compartmental analysis (NCA). The mean terminal half-life (T0) across dose levels was also studied. 1 / 2 The mean clearance (CL) ranged from 47.69 to 78.61 hr, while the mean volume of distribution (Vd, ss) ranged from 0.435 to 0.699 mL / h / kg and 44.3 to 79.0 mL / kg, respectively, both within the typical ranges for marketed therapeutic antibodies. In the 5, 15, or 50 mg / kg IV dose groups, the area under the mean concentration-time curve (AUC) from administration to the last measurable concentration was... last The concentrations were 6.81, 24.9, and 114 h*mg / mL, respectively. No significant gender difference in J30 exposure was observed, but its maximum concentration (C...) was... max ) and AUC last The pharmacokinetic parameters show a slight overproportional trend with the dose. These parameters are summarized in Table 9. Therefore, J30 exhibits pharmacokinetic characteristics similar to those of monoclonal antibodies.

[0205] Table 9. Mean pharmacokinetic parameters after a single J30 IV infusion in cynomolgus monkeys.

[0206]

[0207] Note: All averages are geometric averages. GSD stands for geometric standard deviation coefficient.

[0208] The accompanying toxicokinetics (TK) study of J30 was included in repeated-dose toxicity studies compliant with GLP guidelines (25, 50, and 100 mg / kg dose groups, n=10 per group, administered once weekly for 4 weeks, for a total of 5 doses). Intensive sampling was conducted one week after the first and fourth doses. max and AUC last It showed a slight overproportional trend with the dose. No C was observed. max or AUC last Significant gender differences were observed. After the initial doses of 25, 50, and 100 mg / kg J30, the mean AUC... last The values ​​were 21.6, 54.8, and 126 h*mg / mL, respectively. The TK parameters are summarized in Table 10.

[0209] Table 10. Summary of J30 TK Parameters

[0210]

[0211] *: Arithmetic mean ± SD

[0212] Example 13: Toxicity of J30 in cynomolgus monkeys

[0213] In this GLP compliance study, J30 was administered intravenously once a week for four consecutive weeks (a total of five doses) to evaluate the potential toxicity of J30 in cynomolgus monkeys and to investigate the reversibility, progression, and / or potential delayed effects during the four-week recovery period.

[0214] A total of 20 female and 20 male animals were randomly assigned to four groups (n=5 of each sex in each group). J30 at different dose levels (0, 25, 50, or 100 mg / kg / dose) was administered via IV infusion (over 30 minutes) at a dose volume of 10 mL / kg once a week for four weeks. The study design is shown in Table 11.

[0215] Table 11 Design of a 4-week repeated-dose toxicity study of GLP in cynomolgus monkeys

[0216]

[0217] a The final execution (TS) was scheduled for the 30th day. b The recovery execution (RS) was scheduled for day 58.

[0218] Route of administration: Intravenous infusion (30 min)

[0219] Media control: 20 mM histidine / histidine hydrochloride, 80 mg / mL sucrose, and 0.2 mg / mL polysorbate 80 in water for injection (WFI), pH 5.5.

[0220] M: Male; F: Female.

[0221] J30 was well tolerated when administered intravenously at doses of 25, 50, or 100 mg / kg once weekly for 4 weeks, for a total of 5 doses. No J30-related abnormalities were observed during the administration and recovery periods in terms of body weight, weight gain, food consumption, body temperature, ECG, blood pressure, respiratory function, FOB, clinical observation, ophthalmoscopy, clinical biochemistry, hematology, coagulation, urinalysis, cytokine levels, immunophenotyping, or organ weight. Furthermore, no increase in circulating immune complexes (CIC) was observed.

[0222] At terminal euthanasia, dark red discoloration of the rectal or gastric fundus mucosa was observed in three males administered ≥50 mg / kg. Microscopic examination revealed minimal or slight hemorrhage in the lamina propria. These hemorrhage-related phenomena may be associated with excessive pharmacological effects due to the VEGFA antagonistic function of J30. Because these phenomena were observed only in a small number of animals and were of low severity (minimal or slight), and no related abnormal clinical observations were observed, they were considered non-adverse changes. J30-related histopathological lesions were also found in the heart, lungs, gallbladder, stomach, pancreas, duodenum, ileum, cecum, colon, rectum, kidneys, bladder, ovary, epididymis, brain, sciatic nerve, tongue, cervix, uterus, vagina, or sternum, primarily manifesting as minimal or slight mixed cellular / monocyte infiltration / inflammation or periarteritis. Minimal fibrosis was found in the spleen and skin of a male animal administered 100 mg / kg.

[0223] At the time of euthanasia during the recovery period, no macroscopic changes related to J30 were observed, but microscopic examination revealed minimal mixed cell / monocyte infiltration in the colon, brain, and sciatic nerve, and minimal fibrosis in the lungs, liver, spleen, and cervix. J30-related hemorrhage and periarteritis were completely reversed after the recovery period. J30-related mixed cell / monocyte infiltration / inflammation and fibrinosis were still observed at euthanasia during the recovery period, but due to their lower incidence and milder severity compared to those found at terminal euthanasia, the mixed cell / monocyte infiltration / inflammation was considered partially reversed. None of the above microscopic findings were considered adverse changes.

[0224] Therefore, the highest non-serious toxic dose (HNSTD) / no visible adverse effect level (NOAEL) of J30 was determined to be greater than 100 mg / kg.

[0225] Example 14: Pharmacokinetic (PD) activity of J30 in cynomolgus monkeys

[0226] In the cynomolgus monkey GLP compliance repeated-dose toxicity study described in Example 13, the expression of activation biomarkers (ICOS) and proliferation biomarkers (Ki-67) on CD4 and CD8 T cells was measured using flow cytometry (FCM) to evaluate the T cell activation effect of J30.

[0227] On day 15, in addition to Ki-67+ CD8 T cells, a dose-dependent increase in ICOS+ CD4 / CD8 T cells and Ki-67+ CD4 T cells was observed. Figure 11As shown, on day 15, the median percentage increase in ICOS+ CD8 T cells was 161.89% in the 25 mg / kg group, 225.64% in the 50 mg / kg group, and 279.02% in the 100 mg / kg group. On day 15, the median percentage increase in ICOS+ CD4 T cells was 110.66% in the 25 mg / kg group, 113.45% in the 50 mg / kg group, and 138.92% in the 100 mg / kg group. On day 15, the median percentage increase in Ki-67+ CD4 T cells was 148.09% in the 25 mg / kg group, 132.26% in the 50 mg / kg group, and 229.60% in the 100 mg / kg group. The increase in Ki-67+ CD8 T cells did not show a dose-dependent trend, possibly because it had already reached a plateau at 25 mg / kg. In summary, J30 dose-dependently induced T cell activation in cynomolgus monkeys.

[0228] Table 12. Sequence Information

[0229]

Claims

1. A trispecific antibody comprising a first antigen-binding region binding to PD1, a second antigen-binding region binding to CTLA4, and a third antigen-binding region binding to VEGFa, wherein the first antigen-binding region comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), and wherein: The first VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and the first VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 10, 11 and 12, respectively.

2. The trispecific antibody according to claim 1, wherein the first VH 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: 13, and the first VL 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:

14.

3. The trispecific antibody according to claim 1 or 2, wherein the first antigen-binding region is in the form of scFv.

4. The trispecific antibody according to any one of claims 1-3, wherein the second antigen-binding region comprises a second VH and a second VL, and the second antigen-binding region is in the form of scFv.

5. The trispecific antibody according to any one of claims 1-4, wherein the third antigen-binding region comprises a third VH and a third VL, and the third antigen-binding region is in the form of Fab.

6. The trispecific antibody according to any one of claims 1-5, wherein the first antigen-binding region and the second antigen-binding region are monovalent, and the third antigen-binding region is bivalent.

7. The trispecific antibody according to any one of claims 1-6, wherein the trispecific antibody comprises two heavy chains and two light chains, wherein the two heavy chains comprise heavy chain 1 and heavy chain 2, heavy chain 1 comprising a first antigen-binding region in the form of scFv at its C-terminus, and heavy chain 2 comprising a second antigen-binding region in the form of scFv at its C-terminus.

8. The trispecific antibody of claim 7, wherein each of the two heavy chains comprises the third antigen-binding region VH at its N-terminus, and each of the two light chains comprises the third antigen-binding region VL at its N-terminus.

9. The trispecific antibody according to any one of claims 1-8, wherein the trispecific antibody further comprises a constant region, the constant region comprising a CH3 region, preferably comprising an Fc region.

10. The trispecific antibody according to claim 9, wherein the Fc region has an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

11. The trispecific antibody according to claim 10, wherein the Fc region has a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

12. The trispecific antibody according to any one of claims 9-11, wherein the CH3 region contains a club-and-mortar mutation.

13. The trispecific antibody according to any one of claims 1-12, wherein the second antigen-binding region comprises a second VH and a second VL, and wherein the second VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 15, 16 and 17, respectively, and the second VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 18, 19 and 20, respectively.

14. The trispecific antibody of claim 13, wherein the second VH 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: 21, and the second VL 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:

22.

15. The trispecific antibody according to any one of claims 1-14, wherein the third antigen-binding region comprises a third VH and a third VL, and wherein the third VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 23, 24 and 25, respectively, and the third VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NO: 26, 27 and 28, respectively.

16. The trispecific antibody of claim 15, wherein the third VH 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: 29, and the third VL 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:

30.

17. The trispecific antibody according to any one of claims 1-16, wherein the trispecific antibody comprises two heavy chains and two light chains, wherein heavy chain 1 comprises, from the N-terminus to the C-terminus: the VH, CH1, and Fc regions of the third antigen-binding region and the first antigen-binding region in the form of scFv; heavy chain 2 comprises, from the N-terminus to the C-terminus: the VH, CH1, and Fc regions of the third antigen-binding region and the second antigen-binding region in the form of scFv; and each of the two light chains comprises, from the N-terminus to the C-terminus: the VL region of the third binding region and a light chain constant region.

18. The trispecific antibody according to any one of claims 1-17, wherein the trispecific antibody comprises two heavy chains and two light chains, wherein heavy chain 1 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: 32; heavy chain 2 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: 33; and each of the two light chains 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:

31.

19. A nucleic acid encoding a trispecific antibody according to any one of claims 1-18.

20. A vector comprising the nucleic acid according to claim 19.

21. A host cell comprising the nucleic acid according to claim 19 or the vector according to claim 20.

22. A pharmaceutical composition comprising a trispecific antibody according to any one of claims 1-18, and optionally at least a pharmaceutically acceptable carrier or excipient.

23. The pharmaceutical composition of claim 22, further comprising a second therapeutic agent, optionally selected from the group consisting of free antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, peptides, and small molecule drugs.

24. A method of treating a disease, comprising administering to a subject an effective amount of the trispecific antibody according to any one of claims 1-18, or the pharmaceutical composition according to claim 22 or 23.

25. The method according to claim 24, wherein the disease is cancer, preferably a solid tumor.

26. The method of claim 24 or 25, wherein the solid tumor is selected from the group consisting of: colon cancer, colorectal cancer, prostate cancer, breast cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, liver cancer, hepatocellular carcinoma, gastric cancer, kidney cancer, renal cell carcinoma, cervical cancer, ovarian cancer, and endometrial cancer.

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