CN116375879A - B7-H3 immunoconjugates and uses thereof - Google Patents

Abstract

The invention discloses a B7-H3 immunoconjugate and application thereof. The immunoconjugates comprise an anti-B7H 3 antibody and a cytokine; wherein: the cytokine is IL-2 or a variant thereof. The B7-H3 immunoconjugate of the invention can simultaneously bind with high affinity to B7H3 on the surface of tumor cells and with low affinity to IL-2Rβγ receptor on the surface of killer T cells, and shows good anti-tumor effect both in vivo and in vitro.

Description

B7-H3 immunoconjugates and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a B7-H3 immunoconjugate and application thereof. Furthermore, the invention relates to nucleic acids encoding said immunoconjugates, transformants comprising said nucleic acids and related uses. The application related to the invention comprises the medical application of the immunoconjugate.

Background

B7-H3 (also known as CD 276) is a type I transmembrane glycoprotein, and has a structure very similar to PD-L1, and belongs to the superfamily B7/CD 28. B7-H3 is widely expressed at the transcriptional level (RNA) in lymphoid tissues and non-lymphoid organs, but B7-H3 protein expression is very limited, mainly on activated dendritic cells, monocytes, T lymphocytes, B lymphocytes, NK lymphocytes, but very low in other normal tissues. Recently, B7-H3 has been found to be highly expressed on a variety of solid tumors, such as lung, stomach, pancreas, prostate, kidney, ovary, endometrium, colorectal, liver and breast cancers, and its overexpression is closely related to survival, prognosis or tumor grading. In addition to high expression on tumors, B7-H3 may function like a PD-L1 mediated T cell inhibition signal. B7-H3 has been proposed to have co-stimulatory and co-inhibitory functions, depending on tumor specificity, microenvironment factors and signal intensity. In addition to its role as an immunomodulator, B7-H3 is involved in enhancing metastasis and angiogenesis of cancer.

Since B7-H3 expression is primarily restricted to tumors, B7-H3 is a very important tumor-associated antigen that can be targeted for potential broad-spectrum immunotherapy.

IL-2 (interleukin-2, also known as T Cell Growth Factor (TCGF)), is a multifunctional cytokine produced primarily by activated T cells, particularly CD4+ T helper cells. IL-2 can promote the production of activated immune cells, immune memory cells and immune tolerance. However, in the course of activating immune cells such as T cells, IL-12 activates regulatory T cells (tregs) in addition to effector T cells. The IL-2 receptor consists of three subunits, IL-2Rα (CD 25), IL-2Rβ (CD 122) and IL-2Rγ (CD 132), respectively. The arrangement of these subunits in the receptor determines the response of the receptor to IL-2 signaling. When the three are together (e.g., on Treg cells), the receptor will bind more readily to IL-2 due to the higher affinity of IL-2 for IL-2rα. Whereas IL-2rβ and IL-2rγ are critical for IL-2 signaling, IL-2rα, although not essential for signaling, may confer high affinity binding of IL-2 to the receptor.

IL-2 mediates multiple actions in the immune response through binding to IL-2 receptors on different cells. In one aspect, IL-2 has an immune system stimulating effect and can stimulate proliferation and differentiation of T cells and Natural Killer (NK) cells. Thus, IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. On the other hand, IL-2 can also promote the maintenance of immunosuppressive CD4+CD25+ regulatory T cells (i.e., treg cells) (Fontenot et al, nature Immunol6,1142-51 (2005); D' Cruz and Klein, nature Immunol6,1152-59 (2005)); maloy and Powrie, nature Immunol6,1171-72 (2005)), cause immunosuppression in patients caused by activated Treg cells.

In addition, many years of clinical experience have found that, although high doses of IL-2 can bring about significant clinical efficacy in the treatment of cancers such as melanoma and renal cancer, they can also cause serious drug-related side effects including cardiovascular system toxicity such as vascular leak syndrome and hypotension. Studies have shown that these toxicities are most likely due to the over-activation of lymphocytes (especially T cells and NK cells) by IL-2, which stimulates the release of inflammatory factors. For example, this can cause vascular endothelial cells to contract, increase intercellular spaces, and cause outflow of interstitial fluid, thereby causing vascular leakage side effects.

Another limiting problem with the clinical use of IL-2 is that its extremely short half-life results in dosing difficulties. Because the molecular weight of IL-2 is only 15Kda, the IL-2 can be mainly removed through the filtering action of glomerulus, and the half life of human body is only about 1 hour. To achieve a sufficiently high human exposure, a large dose of IL-2 is clinically infused every 8 hours. However, frequent administration not only places a heavy burden on the patient, but more importantly, large doses of IL-2 infused can result in high peak blood levels (Cmax), which is likely to be another key factor in drug toxicity. Rodrigo Vazquez-Lombardi et al (Nature Communications,8:15373, DOI:10.1038/ncomms 15373) propose to improve the pharmacodynamic properties of interleukins by preparing interleukin 2-Fc fusion, but the fusion protein is expressed in low amounts and is prone to form aggregates.

Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that a combination of IL-2 with certain anti-IL-2 monoclonal antibodies enhances the therapeutic effect of IL-2 in vivo (Kamim ura t a l., JImmiunol 177,306-14 (2006); boyman et al, science 311,1924-27 (2006)). Some modifications of IL-2 molecules have also been proposed. For example, helen R.Mott et al disclose a mutein F42A of human IL-2 with an abolished IL-2Rα binding capacity. Rodrigo Vazquez-Lombardi et al (Nature Communications,8:15373, DOI:10.1038/ncomms 15373) also propose a triple mutant human IL-2 mutein IL-23X with abolished IL-2Rα binding capacity, which has the residue mutations R38D+K43E+E61R at amino acid residue positions 38, 43 and 61, respectively. CN1309705a discloses mutations at positions D20, N88 and Q126 leading to reduced binding of IL-2 to IL-2rβγ. These mutant proteins have drawbacks in terms of pharmacokinetic and/or pharmacodynamic properties, and also have problems of low expression levels and/or poor molecular stability when expressed in mammalian cells. A variety of IL-2 mutations or combinations of mutations are known in the art, and patent WO2021/185361A is incorporated herein as a whole and its use.

Accordingly, there is a need in the art to provide a new molecule with improved properties.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect that in the prior art, T cells which can better target tumor cells and promote T cell differentiation which can kill the tumor cells are lack, and provides a B7-H3 immunoconjugate and application thereof. The immunoconjugate of the invention can simultaneously bind with high affinity to B7H3 on the surface of tumor cells and with low affinity to IL-2Rβγ receptor on the surface of killer T cells, and shows good anti-tumor effect both in vivo and in vitro.

Based on previous studies by the inventors, the inventors tried to make fusion proteins better activate killer T cells by modifying IL-2 to regulate the binding of IL-2 to its receptor while retaining a good targeting ability for B7H3 highly expressed on the surface of tumor cells to bring IL-2 near the tumor cells by targeting of an antibody or antigen binding portion, promote differentiation of killer T cells, and expand immune cells near the tumor cells to achieve antitumor effects. The inventors have screened mutants of IL-2 based on the idea of maintaining IL-2Rα binding and reducing IL-2Rβγ binding; the inventor has unexpectedly found that the BC Loop of the IL-15 has better drug-forming property than the BC Loop which retains the original IL-2, and the immunoconjugate with the characteristics has good anti-tumor effect in vivo and in vitro and has the potential of being a drug for treating B7H3 related diseases and even other tumors.

The invention solves the technical problems by the following technical proposal:

in a first aspect the invention provides an immunoconjugate comprising an anti-B7H 3 antibody and a cytokine or variant thereof; wherein: the cytokine is IL-2 and the anti-B7H 3 antibody is selected from the group consisting of:

(1) The anti-B7H 3 antibody comprises HCDR1 of an amino acid sequence shown as SEQ ID NO. 1, HCDR2 of an amino acid sequence shown as SEQ ID NO. 72 and HCDR3 of an amino acid sequence shown as SEQ ID NO. 3; and/or LCDR1 of the amino acid sequence shown as SEQ ID NO. 4, LCDR2 of the amino acid sequence shown as SEQ ID NO. 5, LCDR3 of the amino acid sequence shown as SEQ ID NO. 6;

(2) The anti-B7H 3 antibody comprises HCDR1 of an amino acid sequence shown as SEQ ID NO. 8, HCDR2 of an amino acid sequence shown as SEQ ID NO. 73 and HCDR3 of an amino acid sequence shown as SEQ ID NO. 10; and/or LCDR1 of the amino acid sequence shown as SEQ ID NO. 74, LCDR2 of the amino acid sequence shown as SEQ ID NO. 12, and LCDR3 of the amino acid sequence shown as SEQ ID NO. 13.

In the invention, the numbering rule of the HCDR1 adopts an AbM numbering rule, and the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 adopt a Kabat numbering rule.

In some embodiments of the invention, the anti-B7H 3 antibody comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein:

(i) The VH comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown as SEQ ID NO. 1 or 8; HCDR2 comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs 2, 7, 9, 14; HCDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 3 or 10; and, a step of, in the first embodiment,

(ii) The VL comprises or consists of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs 4, 11 or 15; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 5 or 12; LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 6 or 13.

In some embodiments of the invention, the anti-B7H 3 antibody comprises or consists of the amino acid sequence shown in SEQ ID NO. 24 and comprises or consists of the amino acid sequence shown in SEQ ID NO. 25.

In some embodiments of the invention, the anti-B7H 3 antibody comprises or consists of the amino acid sequence shown in SEQ ID NO. 26 and comprises or consists of the light chain shown in SEQ ID NO. 27.

In some embodiments of the invention, the anti-B7H 3 antibody comprises or consists of the amino acid sequence shown in SEQ ID NO. 28 and comprises or consists of the light chain shown in SEQ ID NO. 29.

In some embodiments of the invention, the anti-B7H 3 antibody comprises or consists of the amino acid sequence shown in SEQ ID NO. 30 and comprises or consists of the light chain shown in SEQ ID NO. 31.

In some embodiments of the invention, the amino acid sequence of the cytokine mutant is that shown in SEQ ID NO 39, the T mutation at position 3 is A, the F mutation at position 42 is A, the Y mutation at position 45 is A, the L mutation at position 72 is G, the C mutation at position 125 is A or S, the D mutation at position 84 is N, the E mutation at position 95 is Q, the N mutation at position 88 is D, or a combination thereof.

In some preferred embodiments of the invention, the amino acid sequence of the cytokine mutant is that shown in SEQ ID NO 39, wherein the T mutation at position 3 is A, the F mutation at position 42 is A, the Y mutation at position 45 is A, the L mutation at position 72 is G, and the C mutation at position 125 is A; or the amino acid sequence of the cytokine mutant is obtained by mutating the T at the 3 rd position of the amino acid sequence shown as SEQ ID NO. 39 into A, mutating the D at the 84 th position into N, mutating the E at the 95 th position into Q and mutating the C at the 125 th position into S; or the amino acid sequence of the cytokine mutant is obtained by mutating the T at the 3 rd position into A, mutating the N at the 88 th position into D and mutating the C at the 125 th position into S of the amino acid sequence shown as SEQ ID NO 39.

In some embodiments of the invention, the amino acid sequence of BC Loop in the amino acid sequence of the cytokine or mutant thereof is shown in SEQ ID NO. 40.

In some embodiments of the invention, the anti-B7H 3 antibody and cytokine or variant thereof are linked by a linker.

In some preferred embodiments of the invention, the linker is (G ₄ S) ₃ 。

In some preferred embodiments of the invention, the cytokine or variant thereof is linked to the C-terminus of the anti-B7H 3 antibody.

In some preferred embodiments of the invention, the cytokine or mutant thereof is linked to the C-terminus of the knob chain of the anti-B7H 3 antibody.

In some embodiments of the invention, the anti-B7H 3 antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO. 22 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO. 23; the cytokine mutant has an amino acid sequence shown in SEQ ID NO. 76 or 463-591 of SEQ ID NO. 77.

In some preferred embodiments of the invention, the immunoconjugate comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, the first polypeptide chain and the second polypeptide chain being linked by a Knob-in-Hole structure; wherein the first polypeptide chain comprises a first variable region with an amino acid sequence shown as SEQ ID NO. 22 and a first constant region with amino acid sequences shown as SEQ ID NO. 76 and 77, the second polypeptide chain comprises a second variable region with an amino acid sequence shown as SEQ ID NO. 22 and a second constant region with an amino acid sequence shown as SEQ ID NO. 78, and the third polypeptide chain comprises a third variable region with an amino acid sequence shown as SEQ ID NO. 23 and a third constant region with an amino acid sequence shown as SEQ ID NO. 79.

In a second aspect the invention provides an isolated nucleic acid encoding an immunoconjugate according to the first aspect.

In a third aspect the present invention provides a recombinant expression vector comprising a nucleic acid as described in the second aspect.

In a fourth aspect the present invention provides a transformant comprising a nucleic acid as described in the second aspect or a recombinant expression vector as described in the third aspect.

Preferably, the transformant is a cell.

In a fifth aspect the present invention provides a pharmaceutical composition comprising an immunoconjugate according to the first aspect.

A sixth aspect of the invention provides a kit of parts comprising a kit a and a kit B;

wherein the kit a comprises an immunoconjugate according to the first aspect or a pharmaceutical composition according to the fifth aspect; the kit B includes other therapeutic agents.

In some embodiments of the invention, the kit a is administered with the kit B either one after the other or prior to the administration of the kit a; and/or the therapeutic agent is a therapeutic agent having a synergistic effect with the immunoconjugate of kit a.

A seventh aspect of the invention provides the use of an immunoconjugate according to the first aspect, a nucleic acid according to the second aspect, a recombinant expression vector according to the third aspect, a transformant according to the fourth aspect or a pharmaceutical composition according to the fifth aspect for the preparation of a medicament targeting B7-H3 and/or IL-2.

An eighth aspect of the invention provides a method of treating a B7-H3-related disorder, the method comprising administering to a subject in need thereof an effective amount of an immunoconjugate according to the first aspect, a nucleic acid according to the second aspect, a recombinant expression vector according to the third aspect, a transformant according to the fourth aspect, a pharmaceutical composition according to the fifth aspect, or a kit of parts according to the sixth aspect.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

the B7-H3 immunoconjugate of the invention can simultaneously bind with high affinity to B7H3 on the surface of tumor cells and with low affinity to IL-2Rβγ receptor on the surface of killer T cells, and shows good anti-tumor effect both in vivo and in vitro.

Drawings

FIG. 1 shows the results of FACS detection of antibody binding to CHOS cells overexpressing human B7H 3.

FIG. 2 shows the results of antibody ADCC activity assay.

Fig. 3 is an in vivo anti-tumor result of an antibody, wherein a: tumor volume change in tumor-bearing mice, B-C: tumor-bearing mice change in body weight.

FIG. 4 shows the crystal structure of IL-2 and receptor (PDB: 2 ERJ).

FIG. 5 is a molecular pattern diagram of an immunoconjugate of the invention.

FIG. 6 is a schematic diagram showing the results of in vitro functional experiments on anti-B7H3/IL-2m immunoconjugates.

FIG. 7A is a graph showing the effect of 132 and 168 on the antitumor effect of mice; b is a schematic diagram of the effect of 132 and 168 on the body weight of the mice.

FIG. 8A is a graph showing the effect of 176 on the antitumor effect of B16F10 tumors in mice; b is a schematic diagram of the effect of 176 on the body weight of the mice.

Detailed Description

I. Definition of the definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and/or" means when used to connect two or more selectable items, it is understood to mean any one of the selectable items or any two or more of the selectable items.

The terms "comprises" or "comprising" are intended to include the recited element, integer or step, but not to exclude any other element, integer or step. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the instances of the recited elements, integers, or steps in combination. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

Wild-type "interleukin-2" or "IL-2" as used herein refers to a parent IL-2 protein, preferably a naturally occurring IL-2 protein, e.g. a natural IL-2 protein derived from human, mouse, rat, non-human primate, including unprocessed (e.g. without removal of signal peptide) and processed (e.g. with removal of signal peptide) forms, as a template for introducing a mutation or combination of mutations of the invention. A full length native human IL-2 sequence comprising the signal peptide is shown in SEQ ID NO. 41 and the sequence of its mature protein is shown in SEQ ID NO. 39. Furthermore, the expression also includes naturally occurring IL-2 allelic variants and splice variants, isoforms, homologs, and species homologs. The expression also includes variants of native IL-2, e.g., the variants may have at least 95% -99% or more identity to native IL-2 or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions), and preferably have substantially the same IL-2rα binding affinity and/or IL2rβγ binding affinity as the native IL-2 protein. Thus, in some embodiments, wild-type IL-2 may comprise amino acid mutations that do not affect its binding to the IL-2 receptor as compared to the native IL-2 protein, e.g., the native human IL-2 protein (uniprot: P60568) having the mutation C125S introduced at position 125, belongs to the wild-type IL-2 of the invention. An example of a wild-type human IL-2 protein comprising a C125S mutation is shown in SEQ ID NO. 42. In some embodiments, the wild-type IL-2 sequence may have at least 85%, 95%, even at least 96%, 97%, 98%, or 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1 or 2 or 3.

In the present invention, when referring to the IL-2 protein or IL-2 sequence section in the amino acid position, by reference to wild type human IL-2 protein (also called IL-2 WT) amino acid sequence SEQ ID NO:42, was determined. The corresponding amino acid positions on other IL-2 proteins or polypeptides (including full length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO. 42. Thus, in the present invention, unless otherwise indicated, the amino acid position of an IL-2 protein or polypeptide is the amino acid position numbered according to SEQ ID NO. 42. For example, when referring to "F42" it refers to phenylalanine residue F at position 42 of SEQ ID NO. 42, or an amino acid residue aligned at a corresponding position on other IL-2 polypeptide sequences. Meanwhile, for ease of understanding and comparison, when the mutations of the present invention involve a site truncation or deletion of certain specific segments (e.g., the sequence of the B 'C' Loop region (BC Loop), i.e., 11 amino acid residues at positions 73-83 of SEQ ID NO: 42), the amino acid residue numbers outside of that region remain unchanged given the specific mutation region and the manner of mutation thereof, e.g., the sequence of the B 'C' Loop region, i.e., 11 amino acid residues at positions 73-83 of SEQ ID NO:42, is truncated to 7 amino acid residues, 80-83 of the numbers do not reassign, and the amino acid residue position number immediately next to the B 'C' Loop region remains 84. Sequence alignment for amino acid position determination may be performed using Basic Local Alignment Search Tool available from https:// blast.ncbi.lm.nih.gov/blast.cgi, using default parameters.

In this context, when referring to IL-2 muteins, the single amino acid substitutions are described in the following manner: [ original amino acid residue/position/substituted amino acid residue ]. For example, the substitution of lysine at position 35 with glutamic acid may be denoted as K35E. When there can be a number of alternative amino acid substitutions (e.g., D, E) at a given position (e.g., position K35), the amino acid substitution can be expressed as: K35D/E. Accordingly, individual single amino acid substitutions may be joined by a plus sign "+" or "-" to represent a combined mutation at a plurality of given positions. For example, the combined mutations at positions F42A, N88R and S127E can be expressed as: F42A+N88R+S127E, or F42A-N88R-S127E.

IL-2 proteins belong to a short chain class I cytokine family member with four alpha helix bundle (A, B, C, D) structures. The terms "B 'C' Loop" or "B 'C' Loop region" or "B 'C' Loop sequence" are used interchangeably herein to refer to the linking sequence between the B helix and the C helix of the IL-2 protein. By analysis of the crystal structure of IL-2 (e.g., PDB:2 ERJ), the B 'C' loop sequence of an IL-2 protein can be determined. For the purposes of the present invention, the B 'C' loop sequence refers to the sequence of the residue at position 72 and the residue at position 84 linked in the IL-2 polypeptide according to the numbering of SEQ ID NO. 42. In the wild-type IL-2 proteins of SEQ ID NOS.41, 39 and 42, the linker sequence comprises 11 total amino acids A73-R83. Accordingly, in this paper, the term "shortened loop region" or "shortened B 'C' loop region" refers to the wild type IL-2 protein, mutant protein with a length of reduced B 'C' loop sequence, i.e., according to SEQ ID NO:42 numbering, amino acid residues aa72 and aa84 between the connecting sequence length is shortened. "shortened loop region" may be achieved by substitution or truncation of the loop sequence. The substitution or truncation may occur in any region or portion of the B' C loop sequence. For example, the substitution or truncation may be a substitution of the loop region A73-R83 sequence (e.g., to the IL-15B' C loop region) or a truncation from one or more amino acid residues at the C-terminus of the sequence. As another example, the substitution or truncation may be a substitution of the loop Q74-R83 sequence or a truncation from one or more amino acid residues at the C-terminus of the sequence. Following such substitutions or truncations, if desired, single amino acid substitutions may be further introduced in the loop sequence, for example for eliminating glycosylation, and/or back mutations, to further improve the properties of the mutein, for example the pharmaceutical properties. Thus, herein, a mutated shortened B' C loop region can be described by the sequence between the residue at linkage position 72 and the residue at position 84 after introduction of the mutation.

The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody constructs, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, or antibody fragments thereof that exhibit the desired antigen-binding activity. An intact antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, e.g. an antibody naturally occurring in a camel may comprise only heavy chains.

The term "antigen-binding fragment" (used interchangeably herein with "antibody fragment" and "antigen-binding portion") refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody, and binds to an antigen to which the intact antibody binds. Examples of antigen binding fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the Diabodies (dabs); a linear antibody; single chain antibodies (e.g., scFv); single domain antibodies (single domain antibodies);antigen binding fragments of bivalent or bispecific antibodies; camelidae antibodies; and other fragments that exhibit the desired ability to bind antigen (e.g., B7-H3).

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends generally from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al Sequences of Proteins of Immunological Interest,5 ^th As described in Ed.public Health Service, national Institutes of Health, bethesda, MD, 1991.

The term "variable region" or "variable domain" refers to the domain of an antibody that is involved in the heavy or light chain of an antibody binding to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three complementarity determining regions (see, e.g., kit et al Kuby Immunology,6 ^th ed., w.h. freeman and co.91 page (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, VH or VL domains from antibodies that bind to a particular antigen can be used to screen libraries of complementary VL or VH domains, respectively, to isolate antibodies that bind to the antigen, see, e.g., portland et al, j.immunol.150:880-887 (1993); clarkson et al, nature 352:624-628 (1991).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimum overlap region can be determined using at least two of the Kabat, chothia, abM, contact and North methods, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition may be replaced by conserved amino acid residues.

The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., NK cells, neutrophils and macrophages) enable these cytotoxic effector cells to specifically bind antigen-bearing target cells, and subsequently kill the target cells with cytotoxins. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. In order to assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, or ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model. An exemplary assay for assessing ADCC activity is provided in the examples herein.

The term "functional Fc region" refers to an Fc region that possesses the "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors; BCR), etc. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.

As herein describedThe term "linker" as used herein refers to any molecule that enables direct ligation of different portions of a fusion protein. Examples of linkers that establish covalent linkages between different portions of the fusion protein include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol, polypropylene glycol. The term "peptide linker" according to the invention refers to a sequence of amino acids, wherein the sequence connects the amino acid sequence of a first part of a fusion protein to a second part of the fusion protein. For example, a peptide linker may link the IL-2 portion of the fusion protein to the Fc domain or fragment thereof. For example, peptide linkers can also link the antibody to IL-2, such as linking the C-terminus of the antibody's heavy chain to IL-2. Preferably, the peptide linker has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not include predominantly the following amino acid residues: gly, ser, ala or Thr. Useful linkers include glycine-serine polymers including, for example, (GS) n, (GSGGS) n (SEQ ID NO: 43), (G ₄ S)n、(G ₃ S) n and (G) ₄ S) nG, wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Preferably, the linker of the invention is (G ₄ S) _n Where n=1, 2, 3, 4 or 5, preferably 3. Preferably, the linker of the invention is (G ₄ S) ₃ 。

The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating a tumor (e.g., cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response.

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention. For therapeutic or prophylactic purposes, an "effective amount" can be distinguished as a "therapeutically effective amount" and a "prophylactically effective amount". The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: such as the species, size, age and general health of the mammal, the particular disease involved, the extent or severity of the disease, the response of the individual patient, the particular antibody administered, the mode of administration, the bioavailability characteristics of the formulation administered, the selected dosing regimen, and the use of any concomitant therapy.

In one embodiment, an effective amount of a B7-H3 antibody of the invention preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, as compared to a control.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the initially primary transformed cell and the progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term "humanized antibody" refers to antibodies in which antigen binding sites derived from other mammalian species, such as the mouse germline, are grafted onto human immunoglobulin sequences. A humanized antibody is a chimeric molecule that is typically produced using recombinant techniques and may have additional framework region modifications within the human framework sequence. The antigen binding site may comprise the complete variable domain fused to a constant region, or simply comprise the complementarity determining region grafted onto a suitable framework sequence in the variable domain. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs (e.g., 6 CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

The term "immunoconjugate" as used herein refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody or antibody fragment. As described herein, IL-2 molecules can be linked to antibodies through a variety of interactions and in a variety of configurations. For example, fragments of IL-2 and antibody molecules comprising heavy and light chains can form immunoconjugates by dimerization. Preferably, the immunoconjugate of the invention has the structure shown in fig. 5.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.

The term "individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

The term "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

An "isolated nucleic acid encoding an anti-B7-H3 antibody or antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or antigen-binding fragment thereof), including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

Calculation of sequence identity between sequences was performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) j.mol.biol.48:444-453) algorithm (available at http:// www.gcg.com) which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the GAP weights 40, 50, 60, 70 or 80 and the length weights 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum 62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.

The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17).

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.

The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom of cancer, particularly in a subject at risk of cancer.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "subject/patient sample" refers to a collection of tissue or cell samples obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue (e.g., from a fresh, frozen, and/or preserved organ or tissue sample or biopsy or puncture sample); blood or any blood component; body fluids (such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid); cells from any time of gestation or development of a subject.

Antibodies II

Unless otherwise indicated, the terms "B7-H3", "B7H3" and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to a B7/CD28 superfamily member, similar in sequence to the extracellular domain of PD-L1. B7-H3 has 316 amino acids comprising a putative signal peptide consisting of 28 amino acids, an extracellular region consisting of 217 amino acids and a cytoplasmic domain consisting of a transmembrane region and 45 amino acids and has a molecular weight of about 45 kDa to 66kDa. In humans, the extracellular structure of B7-H3 can be either an IgV-IgC-like domain (2 Ig-B7-H3) or an IgV-IgC-like domain (4 Ig-B7-H3) due to exon replication. The sequence of cynomolgus monkey B7-H3 has about 90% homology with its human counterpart.

The term "anti-B7-H3 antibody", "anti-B7-H3", "B7-H3 antibody" or "anti-B7-H3 antibody" as used herein refers to an antibody, or antigen binding fragment thereof, that is capable of binding B7-H3 protein with sufficient affinity. The antibodies may be used as diagnostic and/or therapeutic agents in targeting B7-H3.

In some embodiments, an anti-B7-H3 antibody or antigen-binding fragment thereof of the invention binds B7-H3 (e.g., human or cynomolgus monkey B7-H3) with sufficient affinity, e.g., with an equilibrium dissociation constant (K _D ) Binds to B7-H3, said K _D Less than or equal to 1. Mu.M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10) ^-7 M or less, e.g. 10 ^-7 M to 10 ^-10 M). In some embodiments, B7-H3 is human or cynomolgus monkey B7-H3. In some embodiments, the antibody binding affinity is determined using biological optical interferometry, e.g., in biological optical interferometry, the antibody is at about 1 x 10 ^-7 M or less, about 5X 10 ^-8 M or less, about 1X 10 ^-8 M or less, about 5X 10 ^-9 M or lower K _D About 1X 10 ^-9 M or lower K _D About 1X 10 ^-10 M or lower K _D Binds to human B7-H3.

In some embodiments, the antibodies or antigen binding fragments thereof of the invention bind to cell surface expressed B7-H3.

In some embodiments, the antibodies of the invention, or antigen binding fragments thereof, can induce ADCC effects. In some embodiments, the antibodies of the invention, or antigen binding fragments thereof, can inhibit and/or reduce the growth and/or volume of a tumor in vivo.

In some embodiments, an antibody or antigen-binding fragment thereof that binds B7-H3 of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH and VL comprise a combination selected from the 6 CDRs shown in table a.

In one embodiment of the invention, the amino acid changes described herein include substitutions, insertions or deletions of amino acids. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary permutations are shown in the following table:

In certain embodiments, the substitution occurs in the CDR regions of the antibody. Typically, the resulting variants have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties of the parent antibody that are substantially preserved. Exemplary substitution variants are affinity matured antibodies.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the saccharide attached thereto may be changed. In some applications, modifications that remove unwanted glycosylation sites are useful, for example, to remove fucose moieties to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al (2002) JBC 277:26733). In other applications, galactosylation modifications may be performed to modify Complement Dependent Cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgGl, igG2, igG3, or IgG4Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions. See, for example, U.S. Pat. No. 7,332,581, U.S. Pat. No. 6,737,056; WO 2004/056312 and Shields et al, J.biol.chem.9 (2): 6591-6604 (2001), U.S. Pat. No. 6,194,551, WO 99/51642 and Idusogie et al J.Immunol.164:4178-4184 (2000), U.S. Pat. No. 7,371,826,Duncan&Winter,Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. Cysteine engineered antibodies may be generated as described, for example, in U.S. patent No. 7,521,541.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Nucleic acids of the invention and vectors and host cells comprising the same

The present invention provides nucleic acids encoding any of the above anti-B7-H3 antibodies or antigen binding fragments thereof. Vectors comprising the nucleic acids are also provided. In one embodiment, the vector is an expression vector.

The invention also provides a host cell comprising said nucleic acid or said vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from an E.coli cell, a mammalian cell (e.g., a CHO cell or a 293 cell) or other cell suitable for the preparation of antibodies or antigen binding fragments thereof. In another embodiment, the host cell is prokaryotic. In one embodiment, the host cell is selected from E.coli.

As will be apparent to those of skill in the art, because of the degeneracy of the codons, each antibody or polypeptide amino acid sequence may be encoded by a variety of nucleic acid sequences.

These polynucleotide sequences may be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of sequences encoding antibodies or antigen binding fragments thereof that bind B7-H3, using methods well known in the art.

In one embodiment, one or more vectors comprising a nucleic acid of the invention are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs).

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237,5,789,199 and 5,840,523, also Charlton, methods in Molecular Biology, volume 248 (b.k.c.lo, editions, humana Press, totowa, NJ, 2003), pages 245-254, which describe expression of antibody fragments in e.coli. After expression, the antibodies may be isolated from the bacterial cell paste in the soluble fraction and may be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with a partially or fully human glycosylation pattern. See gerngros, nat. Biotech.22:1409-1414 (2004), and Li et al, nat. Biotech.24:210-215 (2006). Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 HEK or 293 cells, e.g., graham et al, J.Gen. Virol.36:59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA77:216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., yazaki and Wu, methods in Molecular Biology, volume 248 (B.K.C.Lo, ed., humana Press, totowa, NJ), pages 255-268 (2003).

Production and purification of antibody molecules of the invention

In one embodiment, the invention provides a method of preparing an anti-B7-H3 antibody or fragment thereof (preferably an antigen binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody or fragment thereof (preferably an antigen binding fragment), and optionally isolating the antibody or fragment thereof. In a certain embodiment, the method further comprises recovering the anti-B7-H3 antibody or fragment thereof from the host cell.

V. pharmaceutical composition, pharmaceutical preparation and combination product

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-B7-H3 antibody or an immunoconjugate or multispecific antibody thereof and compositions comprising polynucleotides encoding an anti-B7-H3 antibody or an immunoconjugate or multispecific antibody thereof. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.

The pharmaceutical compositions or formulations of the present invention may also be combined with one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other.

Accordingly, in one aspect, the invention also provides a pharmaceutical combination product. In one embodiment, the combination product comprises an antibody, immunoconjugate or multispecific antibody of the invention and a second therapeutic agent formulated in the same pharmaceutical composition or formulation. In another embodiment, the combination product comprises an antibody, immunoconjugate or multispecific antibody of the invention and a second therapeutic agent separately contained in different pharmaceutical compositions or formulations. The second therapeutic agent may be administered prior to, simultaneously with (e.g., in the same formulation or in a different formulation), or after administration of the antibodies of the invention.

The pharmaceutical compositions, formulations and combination products of the present invention may be provided in articles of manufacture for use in the treatment, prevention and/or diagnosis of diseases and/or conditions described herein. The article may comprise a container and a label or package insert. Suitable containers include, for example, bottles, syringes, IV infusion bags, and the like. The container may be made of a variety of materials, such as glass or plastic. In one embodiment, the article of manufacture may comprise (a) a first container containing an antibody or antibody fragment, immunoconjugate or multispecific antibody of the invention; and optionally (b) a second container containing a second therapeutic agent. In addition, the article of manufacture may also contain other materials desirable from a commercial and user standpoint, including buffers, pharmaceutically acceptable diluents, such as sterile water for injection, needles, syringes, syringe pumps, and the like.

VI applications

In one aspect, the invention provides a method of preventing and/or treating a B7-H3 related disease or disorder (e.g., cancer) comprising administering to a subject an effective amount of an immunoconjugate antibody, immunoconjugate, or pharmaceutical composition of the invention.

The subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human. In one embodiment, the subject has or is at risk of having a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy. In other aspects, the invention provides the use of an immunoconjugate antibody, immunoconjugate or pharmaceutical composition in the manufacture or manufacture of a medicament for the prevention and/or treatment of a B7-H3 related disease or disorder as referred to herein.

In some embodiments, an immunoconjugate antibody, immunoconjugate, or pharmaceutical composition or product of the invention delays the onset of the disorder and/or symptoms associated with the disorder.

The immunoconjugates of the invention may be administered by any suitable method, including parenteral, intrapulmonary and intranasal, and, if desired for topical treatment, intralesional. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on whether the administration is short-term or long-term, the administration may be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing at multiple time points, bolus dosing, and pulse infusion.

For the prevention or treatment of a disease, the appropriate dosage of the immunoconjugate of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the immunoconjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one treatment or over a series of treatments.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed in any way as, limiting the scope of the invention, and many modifications will be apparent to those skilled in the art in light of the description herein.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art. A description of these methods can be found, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons, updated 7 in 2008); short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology Greene Pub.associates and Wiley-Interscience; glover, DNA Cloning: A Practical Approach, vol.I & II (IRL Press, oxford, 1985); anand, techniques for the Analysis of Complex Genomes, (Academic Press, new York, 1992); transcription and Translation (b.hames & s.higgins, eds., 1984); perbal, APractical Guide to Molecular Cloning (1984); harlow and Lane, antibodies, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1998) Current Protocols in Immunology Q.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach and w.strober, eds., 1991); annual Review of Immunology; and journal monographs such as Advances in Immunology.

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of hybridoma cells

Immunization of animals

Bal B/c mice (Beijing Veitz) were immunized with recombinant human 4Ig-B7H3 protein (SEQ ID NO: 32) (SINO BIOLOGIAL, cat# 11188-H08H) according to conventional methods, and the recombinant human 4Ig-B7H3 protein (50 ug per mouse) was mixed with TiterMax (Sigma, cat# T2684-1 ML) adjuvant in equal volumes and injected subcutaneously once every two weeks for a total of 5 times.

Cell fusion

When the serum titers meet the requirements, the spleen of the mice is taken according to the conventional method to prepare a B lymphocyte suspension, which is then combined with SP2/0 myeloma cells (ATCC,CRL-1581) is mixed in a ratio of 1:2 to 1:1 and then electrofusion is carried out. Transferring the fused cells from the electrode dish into a 50ml centrifuge tube, diluting the cells with a screening medium (the preparation composition is shown in Table 1) to obtain a cell suspension (concentration 1-2X 10) ⁴ Individual cells/ml). Mu.l of cell suspension was added to each well of a 96-well plate. Fresh screening media was changed on day 5 post-fusion. Positive clones were selected according to the cell growth status, by flow cytometry (FACS) detection after 10 days (or longer) of culture.

Table 1: screening media

High throughput screening of hybridoma cells

Hybridoma cells specifically expressing anti-B7H 3 antibodies were screened by flow cytometry (FACS). Briefly, CHO cells expressing human B7H3 (CHO-huB 7H 3) were counted and diluted to 1×10 ⁶ Mu.l of each cell/ml was then added to each well of the U-bottom 96-well plate, and centrifuged at 500g for 5min to remove the cell culture medium. The culture supernatants of the above hybridoma 96-well plates and a positive control antibody (Enoblituzumab, i.e., MGA271 from macrogenetics) were then added to the U-shaped plates containing CHO cells, respectively, and the cells were resuspended at 100. Mu.l per well and allowed to stand on ice for 30min. The supernatant was removed by centrifugation at 500g for 5min, and the cells were washed 1 time with PBS. Centrifuge 500g for 5min to remove PBS solution. To each well was added 100. Mu.l of FITC-labeled secondary antibody against murine Fab (1:500 diluted in PBS solution), and to the positive control antibody culture wells was added 100. Mu.l of FITC-labeled secondary antibody against human Fab. Incubate on ice for 30min in the dark, centrifuge 500g for 5min to remove supernatant, wash cells 1 time with PBS solution. Cells were then resuspended in 50. Mu.l PBS and FACS detected and screened for positive clones.

The obtained positive clone was subjected to rescreening by using CHO cells (CHO-cynoB 7H 3) expressing cynomolgus monkey B7H3 (SEQ ID NO: 34) in the same manner as described above to obtain 2 hybridoma cells binding to both human B7H3 and cynomolgus monkey B7H 3: 19A2 and 20G5.

The affinity of the obtained 2 hybridoma cell lines to the antigen was measured by using a biofilm thin layer interferometry technique (ForteBio), and the obtained KD values are shown in table 2.

Table 2: forteBio affinity assay results

Subcloning of positive hybridoma cells

The clones were subcloned according to the results of cell binding and affinity assays.

The method comprises the following specific steps: basal medium was obtained by replacing HAT in the screening medium with HT (Gibco, cat # 11067-030) and 200. Mu.l of the basal medium was added to a 96-well plate. The positive hybridoma cells selected from the fusion are selected according to the proportion of about 1 multiplied by 10 ⁵ The density of individual cells/ml, 300 μl per well, was added to the first row of 96-well plates and mixed well. Adding 100 μl of cell suspension of row 1 into row 2, mixing thoroughly, adding 100 μl into next row, repeating the above steps until the last row, and standing for 15min. The counts were observed under a microscope, and the corresponding volumes of 100 cells were added to 20ml of basal medium as described above and plated with 200 μl per well. After two days, the monoclonal wells were judged and labeled by microscopic observation. And (3) when the cell confluence of each hole reaches more than 50%, detecting by adopting the high-throughput FACS screening method, picking out the target positive hole, and freezing the obtained cell clone.

The positive control antibody used in the present invention is MGA271, also known as Enoblituzumab (from macrogeneics US20160264672A 1).

Example 2 preparation of chimeric antibodies

The hybridoma positive clones obtained in example 1 were subjected to the adjustment of the light and heavy chain gene sequences of the antibodies by using molecular biology techniques, and human-mouse chimeric antibodies were constructed using the same.

1.Hybridoma sequencing

Extraction of fresh culture about 5X 10 ⁶ RNA of each hybridoma cell was obtained by reverse transcription using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara)cDNA. The method comprises the following steps:

preparation of reaction System I of Table 3

Table 3:

after incubation at 65℃for 5min, the mixture was rapidly cooled on ice. Reaction system I was added to the following reverse transcription system (Table 4) in a total amount of 20. Mu.l:

table 4: reverse transcription system

After slow mixing, reverse transcription and translation were performed under the following conditions: the cDNA was obtained by cooling at 42℃for 60min to 95℃for 5min and then ice-cooling.

The cDNA was ligated to a T vector, and then heavy and light chain variable regions of an antibody were amplified from the obtained cDNA by PCR using Mighty TA-cloning Kit (Takara), and the PCR reaction system was as shown in Table 5.

Table 5:

the PCR reaction conditions are shown in Table 6.

Table 6:

mu.l of the PCR product obtained by the above PCR reaction was taken, 0.5. Mu.l of pMD20-T vector (Takara), 5. Mu. l Ligation Mighty Mix (Takara) was added, gently mixed, and reacted at 37℃for 2 hours to obtain a ligation product.

Transformed cells:

mu.l of the obtained ligation product was added to E.coli TOP10 competent cells (Tiangen Biochemical Co., ltd., beijing) and incubated on ice for 30min after mixing. After heat shock at 42℃for 90s, the mixture was rapidly cooled on ice for 2min, and 900. Mu.l of LB medium (manufactured and bioengineered (Shanghai) Co., ltd.) was added to the EP tube, followed by shaking culture at 37℃and 220rpm for 1h.3000g was centrifuged for 2min, 800. Mu.l of supernatant was aspirated, and the cells were resuspended in the remaining medium and plated on ampicillin-resistant plates. The cells were incubated overnight at 37℃and the clones were sequenced.

2. Construction of chimeric antibodies

PCR amplification of the anti-B7H 3 antibody VH and VL regions produced by the hybridoma cells of example 1 that have been sequenced: the sequences of the upstream and downstream primers are shown in Table 7 and Table 8.

Table 7: mouse antibody heavy chain variable region (VH) Primer (Primer Mix 1)

After mixing in the above ratios, primer Mix 1 was obtained for PCR amplification of the subsequent VH.

Table 8: light chain variable region (VL) Primer of mouse antibody (Primer Mix 2):

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after mixing in the above ratios, primer Mix 2 was obtained for subsequent PCR amplification of VL.

The PCR system is shown in Table 9.

Table 9:

* For VH chain amplification, primer Mix 1 was applied; for VL chain amplification, primer Mix 2 was applied.

And cutting gel to recover PCR amplified product.

Homologous recombination reaction:

homologous recombination systems are shown in Table 10.

Table 10:

and (3) reacting for 30min at 37 ℃ to obtain a recombinant product. The recombinant product is transformed into TOP10 competence, monoclonal sequencing is selected, clones containing plasmids with correct insertion direction are selected as positive clones, and the positive clones are preserved, so that the recombinant plasmids of the chimeric antibody are obtained. A certain amount of recombinant plasmid is prepared by extraction and used for expressing the antibody.

The present invention provides a total of 2 chimeric antibodies (Ch 19A2 and Ch20G 5) having CDR sequences, light chain variable region and heavy chain variable region sequences identical to the corresponding sequences of the hybridoma cells in tables a-B, the preferred light chain and heavy chain amino acid sequences of which are shown in table 12C.

3. Expression and purification of chimeric antibodies

HEK293 cells (Invitrogen) were passaged in an Expi293 Expression Medium (Gibco, A14351-02) according to the desired transfection volume, and the cell density was adjusted to 1.5X10 one day before transfection ⁶ Individual cells/ml. Cell density on day of transfection was approximately 3X 10 ⁶ Individual cells/ml. Opti-MEM medium (Gibco cat# 31985-070) with a final volume of 1/10 (v/v) was used as transfection buffer, the recombinant expression plasmid constructed above was added, mixed well and filtered with a 0.22 μm filter head for use. Appropriate Polyethylenimine (PEI) (Polysciences, 23966) was added to the plasmid of the previous step (mass ratio of plasmid to PEI 1:3), and after mixing, incubated at room temperature for 10min to obtain a DNA/PEI mixture. The DNA/PEI mixture was gently poured into HEK293 cells and mixed well at 37℃with 8% CO ₂ After 24h of incubation under the conditions of (2) mM final concentration of VPA (Sigma, cat# P4543-100G), and 2% (v/v) of Feed solution (1G/L Phytone Peptone +1G/L Difco Select Phytone), the incubation was continued for 6 days.

After cell culture, the cell culture broth was centrifuged at 13000rpm for 20min, the supernatant was collected, purified by pre-cartridge Hitrap Mabselect Sure (GE, 11-0034-95) according to the manufacturer's instructions, and the concentration was determined. The protein purity was measured by using a gel filtration column SW3000 (TOSOH accession number: 18675) by taking 100. Mu.g of the purified protein and adjusting the concentration to 1mg/mL, and the result showed that a chimeric antibody of high purity was obtained.

EXAMPLE 3 determination of the kinetics of binding of chimeric antibodies of the invention to antigen by thin layer interference techniques of biological films

The equilibrium dissociation constant (KD) of the antibodies of the invention for binding to human B7H3 was determined using biofilm thin layer interferometry (ForteBio). ForteBio affinity assays were performed according to the prior art (Estep, P et al, high throughput solution Based measurement of antibody-antigen affinity and epitope binding. MAbs,2013.5 (2): pages 270-8).

Briefly, AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were equilibrated for 30 minutes offline in assay buffer, then online detection was performed for 60 seconds to establish a baseline, and ForteBio affinity measurements were performed on online loading of purified antibodies obtained as described above onto AHQ sensors (ForteBio). The sensor with the loaded antibodies was then exposed to antigen (including human 4Ig-B7H3, human 2Ig-B7H3 (ACRO, cat. No. B73-H52E 2) and cynomolgus monkey B7H3 (SINO BIOLOGIAL, cat. No. 90806-C02H-50) before the sensor was transferred to an assay buffer for dissociation rate measurement.

The results of the antibody affinity assay are shown in table 11:

table 11: forteBio detects the affinity of antigen-antibody binding (equilibrium dissociation constant KD)

From the above affinity data, we can see that the chimeric antibody obtained by hybridoma has good affinity with human B7H3 protein and also maintains high affinity with cynomolgus monkey B7H 3. The antibodies of the study have higher affinity than MGA271 of the control group.

Example 4 humanization of chimeric antibodies

The chimeric antibody obtained in example 2 was humanized according to a conventional method. Thereby obtaining a humanized antibody: hz20G5, hz19A2, CDR sequences, light chain variable region and heavy chain variable region sequences thereof, amino acid sequences of the light chain and heavy chain are shown in tables 12A-C.

Table 12A: amino acid sequences of CDRs of exemplary antibodies

Table 12B: heavy chain variable region (VH) and light chain variable region (VL) of the exemplified antibodies

Table 12C: exemplary antibody sequences

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EXAMPLE 5 anti-B7H3/IL-2m immunoconjugate design

The immunoconjugates of the invention consist of 1) an antibody against B7H3 and 2) an IL-2 mutant (IL-2 mutein or IL-2 m). Wherein the anti-B7H3 antibody moiety is Hz20G5 obtained in example 4. IL-2m selects a mutation site at the binding interface of the receptor based on the crystal structure 2ERJ (shown in FIG. 4) of the IL-2 and receptor complex to reduce the binding of IL-2 to the receptor; to improve IL-2 formation, the BCLoop (BCL) sequence of IL-2 (A73-R83) was replaced with human IL-15BC Loop. The molecular form of this study is shown in FIG. 5, IL-2m is shown to pass through 3 G ₄ S was linked to Hz20G5 and the heterodimerization of the molecules used the Knob-in-Hole platform.

Table 13: anti-B7H3/IL-2m immunoconjugates

EXAMPLE 6 immunoconjugate preparation

6.1 expression and purification of IL-2 receptor

6.1.1 vector construction

IL-2 receptors IL-2Rα (Uiprot: P01589, aa 22-217) and IL-2Rβ (Uiprot: P14784, aa 27-240) were attached at the C-terminus of the sequence with an avi tag (GLNDIFEAQKIEWHE, SEQ ID NO: 80) that was biotinylated by BirA enzyme and 6 histidine tags (6 XH), respectively, constructed on pTT5 vector (Addgene) for expression of IL-2Rα and IL-2Rβ proteins. The resulting acceptor sequences are shown in SEQ ID NOS 44 and 45, respectively. The constructed plasmids were transferred into HEK293 cells (Invitrogen) to express the prepared proteins, respectively, and plasmid DNA and transfection reagent PEI were first prepared in an ultra clean bench, 3mL of Opti-MEM medium (Gibco cat# 31985-070) was added to a 50mL centrifuge tube, 30. Mu.g of DNA corresponding to the plasmids was added, and the Opti-MEM medium containing the plasmids was filtered using a 0.22 μm filter head, followed by 90. Mu.g PEI (1 g/L) and allowed to stand for 20min. The DNA/PEI mixture was gently poured into 27mL HEK293 cells and mixed well at 37℃with 8% CO ₂ The culture was continued for 6 days under the condition of (C).

Purification of 6.1.2IL-2Rα -His and IL-2Rβ -His proteins

The cell culture broth was centrifuged at 4500rpm for 30min and the supernatant was collected. The supernatant was filtered using a 0.22. Mu.L filter. The nickel column (5mL Histrap excel,GE,17-3712-06) used for purification was immersed in 0.1M NaOH for 2 hours, then rinsed with 5-10 column volumes of ultrapure water to remove lye. The purification column was equilibrated with 5 column volumes of binding buffer (20 mM Tris pH 7.4,300mM NaCl) prior to purification; passing the cell supernatant through a post-equilibration column; removing non-specifically bound heteroproteins by passing 10 column volumes of wash buffer (20mM Tris 7.4,300mM NaCl,10mM imidazole) through the column; the target protein is then eluted with 3-5 column volumes of eluent (20mM Tris 7.4,300mM NaCl,100mM imidazole). The collected proteins were ultrafiltration concentrated and exchanged into PBS (Gibco, 70011-044), then further purified by separation with superdex200 in create (GE, 10/300GL, 10245605), the elution peaks of the monomers were collected, and the equilibration and elution buffer of the column was PBS (Gibco, 70011-044). A100. Mu.g sample of the purified protein was taken and the protein purity was determined using a gel filtration chromatography column SW3000 (TOSOH cat# 18675).

Biotinylation markers for 6.1.3IL-2Rα and IL-2Rβ

The biotin is labeled using an enzyme-catalyzed method as follows: taking appropriate amounts of the above expressed and purified IL-2Rα and IL-2Rβ protein solutions, respectively, adding 1/10 (m/m) of His-birA protein (uniprot: P06709), and simultaneously adding ATP (sigma goods number: A2383-10G) and 5mM MgCl at a final concentration of 2mM ₂ And 0.5mM D-biotin (AVIDITY cat# K0717); incubation at 30℃for 1h, purification by Superdex200 in create (GE, 10/300GL, 10245605) and removal of excess biotin and His-birA; the purified samples were validated by Fortebio's Streptavidin (SA) sensor (PALL, 18-5019) to confirm that the biotin labeling was successful.

6.2 preparation of immunoconjugates

Vectors containing fusion protein genes encoding immunoconjugates as shown in the above table were transferred into HEK293 cells for expression using transient transfection methods. Plasmid DNA and transfection reagent PEI were first prepared in an ultra clean bench, 3mL of Opti-MEM medium (Gibco cat# 31985-070) was added to a 50mL centrifuge tube, 30. Mu.g of DNA corresponding to the plasmid was added, the Opti-MEM medium containing the plasmid was filtered using a 0.22 μm filter, followed by 90. Mu.g PEI (1 g/L) and allowed to stand for 20min. The DNA/PEI mixture was gently poured into 27mL HEK293 cells and mixed well at 37℃with 8% CO ₂ The culture was continued for 6 days under the condition of (C).

Affinity purification: the cells were centrifuged at 13000rpm for 20min, the supernatant was collected and purified by pre-cartridge Hitrap Mabselect Sure. The operation is as follows: before purification, the packed column is equilibrated with 5 column volumes of equilibration liquid (0.2M Tris,0.15M NaCl,pH7.2); passing the collected supernatant through a column, and then washing the packed column with a balancing solution with the volume of 10 times of the column volume to remove the non-specific binding protein; flushing the packing with 5 column volumes of elution buffer (0.1M sodium citrate,pH 3.5) and collecting the eluate; mu.L of Tris (2M Tris) was added per 1mL of eluate, and the eluate was concentrated by ultrafiltration into PBS buffer, and the concentration and purity were measured.

Ion exchange purification: the heterodimeric molecules in the immunoconjugate were isolated by ion exchange chromatography, removing homodimeric impurities.

EXAMPLE 7 affinity assay of immunoconjugates

The equilibrium dissociation constants (equilibrium dissociation constant, KD) for antibodies of the invention to bind human B7H3, IL-2Rα and IL-2Rβ were determined using biofilm interferometry techniques (Biolayer Interferometry, BLI). The BLI method affinity assay was performed according to the known method (Estep, P et al, high throughput solution Based measurement of antibody-antigen affinity and epitope binding. MAbs,2013.5 (2): pages 270-8).

Half an hour before the start of the experiment, a suitable number of AHC (18-5060, fortebio) or SA sensors (PALL, 18-5019) were immersed in SD buffer (1 XPBS, BSA 0.1%, tween-20.05%) depending on the number of samples.

mu.L of SD buffer, 100nM of fusion protein molecules (132, 168 and 176), 100nM of human B7H3 (cat# 11188-H08H), 100nM of human IL-2Rα -Biotin (prepared in example 6) and 100nM of human IL-2Rβ -Biotin (prepared in example 6) were added to 96-well black polystyrene half-microwell plates (Greiner, 675076), respectively. Detection was performed using Fortebio Octet Red96e, with sensor positions selected based on sample position layout. The affinity of B7H3 was determined by binding to B7H3 after antibody immobilization by AHC sensor; the affinity for IL-2 receptor was determined by binding to IL-2 immunoconjugates (132, 168 and 176) after immobilization of the receptor by SA sensor. The instrument set parameters are as follows: the operation steps are as follows: baseline, antibody Loading-1 nm, baseline, antigen Association and Association; the run time of each step was dependent on the sample binding and dissociation speed, with a rotational speed of 1000rpm and a temperature of 30 ℃. KD values were analyzed using ForteBio Octet analysis software.

The results of the experiments are shown in tables 14 to 16.

Table 14: immunoconjugates and recombinant human B7H3 affinity

Sample name	KD(M)	Kon(1/ms)	Kdis(1/s)
				132	3.11E-10	6.78E+05	2.11E-04
168	4.52E-10	6.86E+05	3.10E-04
				176	8.02E-10	7.01E+05	5.62E-04

Table 15: immunoconjugates and recombinant human IL-2Rα affinity

*N.B.＝No Binding

Table 16: immunoconjugates and recombinant human IL-2 Rbeta affinity

*N.B.＝No Binding

Example 8 in vitro Activity assay of immunoconjugates

IL-2 binds to CTLL2 cell surface IL-2 receptors (IL-2Rα, IL-2Rβ and IL-2Rγ) and activates the CTLL2 JAK-STAT signaling pathway, triggering a reporter gene signal.

The experimental method comprises the following steps:

1. configuring an Assay Medium:1% MEM NEAA (Gibco, 11140-050), 10% FBS (PEAK, PS-FB 1) and 89% IMDM (Gibco, 12440-053).

2. CTLL2 cells were washed 2 times with Assay Medium.

3. CTLL2 (or CTLL2-hPD-1, promega, CS 201805) cell densities were adjusted with a Medium containing 0.4ng/mL rhIL2 (R & D, 202-IL) Assay Medium, and Medium 96-well white cell culture plates (NUNC) were plated with 50000 cells per well.

4. Edge wells were plated with equal volume Assay Medium, 5% CO ₂ Starvation treatment is carried out at 37 ℃ for 18-20h.

5. Diluted immunoconjugates to be tested were added to the cell plates, 5% CO, respectively ₂ Incubate at 37℃for 6h.

6. The plates were removed and equilibrated at room temperature for 15-20min, equal volumes of Luciferease assay system reagent (Bio-Glo) were added to each well, incubated at room temperature for 5-15min, and read by an enzyme-labeled instrument (Molecular Devices).

As shown in FIG. 6, IL-2m at 168 and 176 has a strong STAT5 activation signal on CTLL2 cells, which is significantly stronger than that of control molecule 132.

EXAMPLE 9 in vivo efficacy experiments of immunoconjugates

To demonstrate the efficacy of the novel engineered immunoconjugates in vivo, the anti-tumor efficacy of the immunoconjugates of the invention (132 and 168) was determined by inoculating C57 mice with MC38 cell (mouse colon cancer cell line) overexpressing human B7H3 molecules. SPF-grade female C57 mice (purchased from Experimental animal technologies Inc. of Toril, beijing) were used for the experiment, and the certification number was No.1100112011005928.

MC38-B7H3 cells are constructed primarily by the following method: human B7H3 sequence (Uniprot ID: Q5ZPR 3) was inserted into pWPT-GFP vector (Plasmid # 12255-Addgene) and transferred into 293T cells together with the corresponding helper vector to form lentiviruses. After infection of MC38 cells with lentivirus, B7H3 positive clones were isolated by flow cytometry and the final cells obtained were MC38-B7H3 cells.

MC38-B7H3 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and MC38-B7H3 cells were resuspended in PBS (1X) to a cell concentration of 15X 10 ⁶ Cell suspension per mL. 0.2mL of the cell suspension was inoculated subcutaneously into the right flank region of C57 mice on day 0 to establish a MC38-B7H3 tumor-bearing mouse model.

Tumor volumes of each mouse were measured 7 days after tumor cell inoculation and were grouped (7 mice per group) and the dosing and modes are shown in table 17.

Table 17: grouping, dosing and mode of in vivo experiments

Group of	Dosage for administration	Number of administrations	Administration mode
				h-IgG*	10mg/kg	QW×3	Abdominal cavity injectionRadiation
132	3mg/kg	QW×3	Intraperitoneal injection
				132	10mg/kg	QW×3	Intraperitoneal injection
168	3mg/kg	QW×3	Intraperitoneal injection
				168	10mg/kg	QW×3	Intraperitoneal injection

* : h-IgG was isotype control antibody, purchased from Equitech-Bio, lot 161206-0656.

The in vivo doses of h-IgG were 10mg/kg, and the 132 and 168 molecules were administered in two doses, one 3mg/kg and the other 10mg/kg, 3 times weekly (QW. Times.3). Mice tumor volume and body weight were monitored 2 times per week on days 7, 14, 21 after MC38-B7H3 cell inoculation, respectively, and ended after 24 days of monitoring as shown in a of fig. 7. The relative tumor inhibition (TGI) was calculated on day 24 post inoculation as follows: TGI% = 100% × (control tumor volume-treated tumor volume)/(control tumor volume-control tumor volume pre-dose tumor volume). Tumor volume determination: the maximum axis (L) and the maximum axis (W) of the tumor were measured by vernier calipers, and the tumor volume was calculated according to the following formula: v=l×w ² /2. CollectingBody weight was measured with an electronic balance.

Tumor inhibition results are shown in table 18: on day 24 post-inoculation, tumor inhibition rates of 132,3mg/kg, 10mg/kg and 168,3mg/kg, 10mg/kg were 45%, 75% and 80%, 88%, respectively, compared to the h-IgG,10mg/kg group. The results of simultaneous detection of the body weight of the mice (B of fig. 7) showed that we observed that 132 had a significant effect on the change in body weight of the mice and exhibited a dose effect, with a weight loss of more than 10% occurring in 4 out of 7 mice in the 10mg/kg group, with two mice eventually dying. In contrast, no significant weight loss occurred and none of the mice died in group 168. 168 thus showed more excellent potency and safety than 132 at the same dose.

Table 18: tumor inhibition on day 24

Group of	Tumor volume (mm) 3 )	Tumor inhibition rate (%)	Death of mice	Maximum weight loss (%)
					h-IgG	998.31	N/A*	0/7	N/A
132,3mg/kg	578.03	45	0/7	1.63
					132,10mg/kg	302.62	75	2/7	9.05
168,3mg/kg	255.97	80	0/7	N/A
					168,10mg/kg	184.06	88	0/7	2.85

*：N/A＝Not Available

To further demonstrate the in vivo efficacy of the immunoconjugate, the anti-tumor efficacy of the 176 immunoconjugate of the invention was determined by inoculating C57 mice with PD1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC: CRL-6475). SPF-grade female C57 mice (purchased from Experimental animal technologies Co., ltd., beijing, vitre.) were used for the experiment, and the certification number was No.202103331Abzz0619000743.

B16F10 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation, and B16F10 cells were resuspended in PBS (1X) to a cell concentration of 2.5X10 ⁶ Cell suspension per mL. On day 0, 0.2mL of the cell suspension was inoculated subcutaneously into the right flank region of C57 mice to establish a B16F10 tumor-bearing mouse model.

Tumor volumes of each mouse were measured 8 days after tumor cell inoculation and were grouped (7 mice per group) and the dosing and modes are shown in table 7.

Table 19: grouping, dosing and mode of in vivo experiments

Group of	Dosage for administration	Number of administrations	Administration mode
				h-IgG*	10mg/kg	QW×2	Intraperitoneal injection
176	3mg/kg	QW×2	Intraperitoneal injection

* : h-IgG was isotype control antibody, purchased from Equitech-Bio, lot 161206-0656.

The doses of h-IgG and 176 are shown in the above table, with the dosing frequency being 2 times per week (QW 2). Mice tumor volume and body weight were monitored 2 times per week on days 8, 15 after B16F10 cell inoculation, respectively, and ended after 22 days of monitoring as shown in fig. 8 a. Since mice die from tumors at an early stage in the group, we calculated the relative tumor inhibition (TGI%) on day 15 post-inoculation, and calculated the formulaThe formula is as follows: TGI% = 100% × (control tumor volume-treated tumor volume)/(control tumor volume-control tumor volume pre-dose tumor volume). Tumor volume determination: the maximum axis (L) and the maximum axis (W) of the tumor were measured by vernier calipers, and the tumor volume was calculated according to the following formula: v=l×w ² /2. Body weight was measured using an electronic balance.

The tumor growth curves are shown in table 20: on day 15 post-inoculation, 176 tumor inhibition was 79% compared to the h-IgG,10mg/kg group. The results of simultaneous detection of the body weights of the mice (B of fig. 8) showed no significant difference in body weights of the mice at day 29 after inoculation.

Table 20: tumor inhibition on day 15

Group of	Tumor volume (mm) 3 )	Tumor inhibition rate (%)
			h-IgG	712.70	N/A*
176	206.95	79

* : n/a=not Available, not Available.

Claims (12)

1. An immunoconjugate comprising an anti-B7H 3 antibody and a cytokine;

wherein: the cytokine is IL-2 or a mutant thereof, and the anti-B7H 3 antibody is selected from the group consisting of:

(1) The anti-B7H 3 antibody comprises HCDR1 of an amino acid sequence shown as SEQ ID NO. 1, HCDR2 of an amino acid sequence shown as SEQ ID NO. 72 and HCDR3 of an amino acid sequence shown as SEQ ID NO. 3; and/or LCDR1 of the amino acid sequence shown as SEQ ID NO. 4, LCDR2 of the amino acid sequence shown as SEQ ID NO. 5, LCDR3 of the amino acid sequence shown as SEQ ID NO. 6;

(2) The anti-B7H 3 antibody comprises HCDR1 of an amino acid sequence shown as SEQ ID NO. 8, HCDR2 of an amino acid sequence shown as SEQ ID NO. 73 and HCDR3 of an amino acid sequence shown as SEQ ID NO. 10; and/or LCDR1 of the amino acid sequence shown as SEQ ID NO. 74, LCDR2 of the amino acid sequence shown as SEQ ID NO. 12, and LCDR3 of the amino acid sequence shown as SEQ ID NO. 13.

2. The immunoconjugate of claim 1, wherein the anti-B7H 3 antibody comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

(i) The VH comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown as SEQ ID NO. 1 or 8; HCDR2 comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs 2, 7, 9, 14; HCDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 3 or 10; and, a step of, in the first embodiment,

(ii) The VL comprises or consists of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs 4, 11 or 15; LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 5 or 12; LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 6 or 13.

3. The immunoconjugate of claim 2, wherein the anti-B7H 3 antibody is selected from the group consisting of:

1) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 24, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 25;

2) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 26, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 27;

3) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 28, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 29;

4) Comprising or consisting of the amino acid sequence shown in SEQ ID NO. 30, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO. 31.

4. The immunoconjugate of claim 2, wherein the cytokine mutant has an amino acid sequence of SEQ ID No. 39, wherein the T mutation at position 3 of the amino acid sequence is a, the F mutation at position 42 is a, the Y mutation at position 45 is a, the L mutation at position 72 is G, the C mutation at position 125 is a or S, the D mutation at position 84 is N, the E mutation at position 95 is Q, the N mutation at position 88 is D, or a combination thereof;

preferably, the amino acid sequence of the cytokine mutant is obtained by mutating the T at the 3 rd position into A, mutating the F at the 42 th position into A, mutating the Y at the 45 th position into A, mutating the L at the 72 th position into G and mutating the C at the 125 th position into A of the amino acid sequence shown in SEQ ID NO 39; or the amino acid sequence of the cytokine mutant is obtained by mutating the T at the 3 rd position of the amino acid sequence shown as SEQ ID NO. 39 into A, mutating the D at the 84 th position into N, mutating the E at the 95 th position into Q and mutating the C at the 125 th position into S; or the amino acid sequence of the cytokine mutant is obtained by mutating the T at the 3 rd position into A, mutating the N at the 88 th position into D and mutating the C at the 125 th position into S of the amino acid sequence shown as SEQ ID NO 39.

5. The immunoconjugate of claim 4, wherein the amino acid sequence of BC Loop in the amino acid sequence of the cytokine or mutant thereof is shown in SEQ ID No. 40.

6. The immunoconjugate of any one of claims 1 to 5, wherein the anti-B7H 3 antibody and cytokine or mutant thereof are linked by a linker;

preferably, the linker is (G ₄ S） ₃ The method comprises the steps of carrying out a first treatment on the surface of the And/or the cytokine or mutant thereof is linked to the C-terminus of the heavy or light chain of the anti-B7H 3 antibody; preferably at the C-terminus of the knob chain of said anti-B7H 3 antibody;

more preferably, the anti-B7H 3 antibody has a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 22 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 23; the cytokine mutant has an amino acid sequence shown in SEQ ID NO. 76 or 463-591 of SEQ ID NO. 77;

even more preferably, the bifunctional fusion protein comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, said first and second polypeptide chains being linked by a Knob-in-Hole structure; wherein the first polypeptide chain comprises a first variable region with an amino acid sequence shown as SEQ ID NO. 22 and a first constant region with amino acid sequences shown as SEQ ID NO. 76 and 77, the second polypeptide chain comprises a second variable region with an amino acid sequence shown as SEQ ID NO. 22 and a second constant region with an amino acid sequence shown as SEQ ID NO. 78, and the third polypeptide chain comprises a third variable region with an amino acid sequence shown as SEQ ID NO. 23 and a third constant region with an amino acid sequence shown as SEQ ID NO. 79.

7. An isolated nucleic acid encoding the immunoconjugate of any one of claims 1 to 6.

8. A recombinant expression vector comprising the nucleic acid of claim 7.

9. A transformant comprising the nucleic acid of claim 7 or the recombinant expression vector of claim 8;

preferably, the transformant is a cell.

10. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 6.

11. A packaged medicine box, which is characterized by comprising a medicine box A and a medicine box B;

wherein the kit a comprises the immunoconjugate of any one of claims 1 to 6 or the pharmaceutical composition of claim 10; the kit B includes other therapeutic agents;

preferably, the administration time of the medicine box A and the medicine box B is not different from each other or the medicine box A is firstly administered; and/or the therapeutic agent is a therapeutic agent having a synergistic effect with the immunoconjugate of kit a.

12. Use of an immunoconjugate according to any one of claims 1 to 6, a nucleic acid according to claim 7, a recombinant expression vector according to claim 8, a transformant according to claim 9 or a pharmaceutical composition according to claim 10 for the preparation of a medicament for targeting B7-H3 and/or IL-2.

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