阅读说明：本技术 用于治疗egfr阳性癌症的t细胞重定向双特异性抗体 (T cell redirecting bispecific antibodies for the treatment of EGFR-positive cancer ) 是由 R·利西拉 C·斯图茨 S·布莱因 于 2018-04-24 设计创作，主要内容包括：本发明涉及同时结合CD3和EGFR的双特异性抗体。发明人已经证明这类抗体可用于治疗EGFR肿瘤，其通过重定向T细胞并在活化的T细胞和表达EGFR的肿瘤细胞之间形成免疫突触，从而导致表达EGFR的肿瘤细胞的杀伤水平提高。特别地，本发明涉及选自以下的CD3xEGFR双特异性抗体：CD3xEGFR_SF1(SEQ ID NO：4、5和6)、CD3xEGFR_SF3(SEQ ID NO：7、2和8)、CD3xEGFR_SF4(SEQ ID NO：4、5和9)、CD3xEGFR_SD1(SEQ ID NO：1、2和10)和CD3xEGFR_SD2(SEQ ID NO：11、10和2)。(The present invention relates to bispecific antibodies that bind both CD3 and EGFR. The inventors have demonstrated that such antibodies are useful for treating EGFR tumors by redirecting T cells and forming immunological synapses between activated T cells and EGFR-expressing tumor cells, resulting in increased levels of killing of EGFR-expressing tumor cells. In particular, the invention relates to a CD3xEGFR bispecific antibody selected from: CD3xEGFR _ SF1(SEQ ID NOS: 4, 5 and 6), CD3xEGFR _ SF3(SEQ ID NOS: 7, 2 and 8), CD3xEGFR _ SF4(SEQ ID NOS: 4, 5 and 9), CD3xEGFR _ SD1(SEQ ID NOS: 1, 2 and 10) and CD3xEGFR _ SD2(SEQ ID NOS: 11, 10 and 2).)

1. A CD3xEGFR bispecific antibody that binds to an epitope on CD3 epsilon and EGFR.

2. The CD3xEGFR bispecific antibody of claim 1 comprising at least one FAB and at least one scFv moiety.

3. The CD3xEGFR bispecific antibody according to claim 2, wherein the at least one FAB and one scFv moiety are linked to each other.

4. The CD3xEGFR bispecific antibody according to any one of claims 1 to 3, selected from the group comprising CD3xEGFR _ SF1(SEQ ID NOS: 4, 5 and 6), CD3xEGFR _ SF3(SEQ ID NOS: 7, 2 and 8), CD3xEGFR _ SF4(SEQ ID NOS: 4, 5 and 9), CD3xEGFR _ SD1(SEQ ID NOS: 1, 2 and 10) and CD3xEGFR _ SD2(SEQ ID NOS: 11, 10 and 2).

5. A CD3xEGFR bispecific antibody according to any one of claims 1-4 for use as a medicament.

6. The CD3xEGFR bispecific antibody according to any one of claims 1-4, for use in the treatment of EGFR expressing cancer.

7. The CD3xEGFR bispecific antibody according to claim 6, wherein the EGFR expressing cancer further comprises one or more KRAS or B-Raf mutations.

8. An antibody or fragment thereof that binds to domain 4 of human EGFR comprising heavy and light variable sequences selected from or derived from: SEQ ID NO: 23 and 24, SEQ ID Ns: 25 and 26, SEQ ID NO: 31 and 33, SEQ ID NO: 32 and 34, SEQ ID NO: 36 and 38, SEQ ID NO: 37 and 39.

Technical Field

The present invention relates to bispecific antibodies that bind both CD3 and EGFR. The inventors have demonstrated that such antibodies are useful for treating EGFR tumors by redirecting T cells and forming immunological synapses between activated T cells and EGFR-expressing tumor cells, resulting in increased levels of killing of EGFR-expressing tumor cells.

Background

It has been demonstrated that the use of anti-EGFR monoclonal antibodies (mabs) to target Epidermal Growth Factor Receptor (EGFR), which is overexpressed by many cancer cells of epithelial origin, has been shown to be useful in inhibiting the growth of such tumor cells, leading to positive clinical outcomes.

Cancer immunotherapy or immunooncology is a fourth anti-tumor approach that has progressed over a period of time after undergoing some encouraging studies and other compelling data regarding its clinical efficacy.

However, the clinical response of patients treated with anti-EGFR mabs is variable and may reflect variability of EGFR expression, signaling in tumor cells, adaptive mechanisms of cancer cells for evading therapy, or some combination of all of these factors.

One well-elucidated mechanism by which cancer cells develop resistance to anti-EGFR mAb treatment is by mutating a Kirsten Ras (KRAS) oncogene homolog from the mammalian ras gene family. Somatic KRAS mutations are found in leukemia, colorectal, pancreatic and lung cancers. KRAS mutations predictive of the approved anti-EGFR mAb therapeutic panitumumab

And cetuximab

The response in colorectal cancer is very poor.Studies have shown that patients with tumors expressing mutated forms of the KRAS gene do not respond to cetuximab or panitumumab. The emergence of KRAS mutations is a common driver of acquired resistance of colorectal and other cancers to anti-EGFR mAb therapeutics.

Disclosure of Invention

To address the problems associated with EGFR cancer treatment, the inventors have generated a new set of anti-tumor drugs suitable for treating EGFR-overexpressing cancers and overcoming the problems of existing treatments.

The present invention relates to bispecific antibodies that bind to CD3 epsilon and an epitope on EGFR.

Wherein the CD3 epsilon binding agent is preferably SP34 or OKT3 or derived therefrom.

Wherein the EGFR binder is preferably panitumumab and cetuximab.

According to the present invention, a CD3xEGFR bispecific antibody comprises at least one FAB and one scFv binding moiety.

In particular, the invention relates to binding moieties from protein-based target specific binding molecules such as, but not limited to, antibodies, DARPins, Fynomers, Affimers, variable lymphocyte receptors, anticalins, nanofitin, variable neoantigen receptors (VNARs).

In particular, the binding moiety is taken from or derived from an antibody, e.g., Fab '-SH, Fd, Fv, dAb, F (ab') 2, scFv, Fcabs, bispecific single chain Fv dimers, diabodies, triabodies. In a preferred embodiment, the agonist comprises a binding moiety taken from or derived from a Fab, ScFv and dAb.

According to the present invention, a CD3xEGFR bispecific antibody comprises at least one FAB and one scFv moiety linked to each other.

In particular, the binding moieties may be genetically fused to a scaffold comprising the same or different antibody Fc or portions thereof. According to this aspect of the invention, a first full length antibody, e.g. IgG, may form the basis of a CD3xEGFR bispecific antibody according to the invention and a second set of binding moieties may be grafted onto the starting antibody according to the invention.

Preferably, the two binding moieties are linked such that the second binding moiety is distal to the variable portion of the immunoglobulin heavy chain.

Alternatively, the two binding moieties are linked such that the second binding moiety is proximal to the variable portion of the immunoglobulin heavy chain.

Preferably, the two binding moieties are linked such that the second binding moiety is distal to the variable portion of the immunoglobulin light chain.

Alternatively, the two binding moieties are linked such that the second binding moiety is proximal to the variable portion of the immunoglobulin light chain.

According to the invention, the two linked binding moieties may be separated by a peptide linker.

According to the invention, the CD3xEGFR bispecific antibody is selected from the group comprising CD3xEGFR _ SF1(SEQ ID NOS: 4, 5 and 6), CD3xEGFR _ SF3(SEQ ID NOS: 7, 2 and 8), CD3xEGFR _ SF4(SEQ ID NOS: 4, 5 and 9), CD3xEGFR _ SD1(SEQ ID NOS: 1, 2 and 10) and CD3xEGFR _ SD2(SEQ ID NOS: 11, 10 and 2).

According to another aspect of the invention, there is provided an antibody or fragment thereof that binds to domain 4 of human EGFR, said antibody or fragment thereof comprising a heavy variable sequence and a light variable sequence selected from the group consisting of: SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 31 and 33, SEQ ID NO: 32 and 34, SEQ ID NO: 36 and 38, SEQ ID NO: 37 and 39 or derived therefrom.

The invention also relates to the use of a CD3xEGFR bispecific antibody according to the invention as a medicament.

The invention also relates to the use of a CD3xEGFR bispecific antibody according to the invention as a medicament for the treatment of cancer or other diseases characterized by or exacerbated by overexpression of EGFR.

The invention also relates to a method of treating a patient suffering from cancer comprising administering to the patient an effective amount of a CD3xEGFR bispecific antibody.

The invention also relates to a method of treating a patient suffering from cancer comprising administering to the patient an effective amount of a CD3xEGFR bispecific antibody and one or more other agents, such as a small molecule or a biologic drug, to further modulate the patient's immune system. Examples of such agents include anti-PD-1 antibodies and anti-tumor small molecules, such as multi-kinase inhibitors.

Furthermore, the invention relates to the co-administration of a CD3xEGFR bispecific antibody according to the invention and a further drug to a patient, wherein the further drug has a synergistic or additive effect.

According to another aspect of the present invention, there is provided a method of treating EGFR-expressing cancer by administering to a patient in need thereof a therapeutic amount of a CD3xEGFR bispecific antibody according to the invention.

According to another aspect of the invention, there is provided a CD3xEGFR bispecific antibody according to the invention for use as a medicament.

According to another aspect of the invention, there is provided a CD3xEGFR bispecific antibody according to the invention for use in the treatment of EGFR expressing cancer.

According to another aspect of the invention, the EGFR expressing cancer further comprises one or more KRAS or B-Raf mutations provided.

Unless defined otherwise, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques described herein relating to cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly done in the art or as described herein. The foregoing techniques and processes are generally performed according to conventional methods well known in the art, and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, and the laboratory procedures and techniques described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery and patient treatment.

The basic antibody building block is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region, primarily responsible for effector (effector) function. Generally, antibody molecules obtained from humans relate to any of IgG, IgM, IgA, IgE and IgD, which differ from each other in their heavy chain properties. Certain classes also have subclasses (also referred to as isotypes), such as lgG1, lgG2, and the like. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules comprising only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the Complementarity Determining Regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. Mabs comprise an antigen binding site that immunoreacts with a particular epitope of an antigen and are characterized by a unique binding affinity for it.

The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in binding to an antigen. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") region of the heavy ("H") and light ("L") chains. Three highly divergent stretches (called "hypervariable regions") within the V regions of the heavy and light chains are inserted between the more conserved flanking segments called "framework regions" or "FRs". Thus, the term "FR" refers to amino acid sequences that naturally occur between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs". The allocation of amino acids to each domain is defined according to the Kabat sequence of proteins of immunological interest ((National Institutes of health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J.mol.biol.196: 901-917(1987), Chothia et al, Nature 342: 878-883 (1989)).

The single domain antibody (sdAb) fragment portion of the fusion proteins of the present disclosure is interchangeably referred to herein as a targeting polypeptide.

As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof or a T cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, usually having specific three-dimensional structural characteristics as well as specific charge characteristics. An antibody can be said to specifically bind an antigen when the dissociation constant is ≦ 1mM, e.g., ≦ 1 μ M in certain embodiments (such as ≦ 100nM, ≦ 10nM, or ≦ 1 nM).

As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for an immunoglobulin. The strength or affinity of an immunological binding interaction may be expressed in terms of the dissociation constant (KD) of the interaction, where a smaller KD indicates a greater affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rate of antigen binding site/antigen complex formation and dissociation, where the rate depends on the concentration of the complexing partner, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on rate constant" (kon) and the "off rate constant" (koff) can be determined by calculating the concentration and the actual rate of association and dissociation (see Nature 361: 186-87 (1993)). The koff/kon ratio eliminates all parameters that are independent of affinity and is equal to the dissociation constant KD (see generally Davies et al, (1990) Annual Rev Biochem 59: 439-473). An antibody of the invention is said to specifically bind an antigen when the equilibrium binding constant (KD) is 1mM or less, in some embodiments 1. mu.M or less, 100nM or less, 10nM or less, or 100pM or less, to about 1 pM. Measured by assays such as radioligand binding assays, Surface Plasmon Resonance (SPR), flow cytometry binding assays or similar assays known to those skilled in the art.

The term "isolated protein" as used herein refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which, due to its advantages or origin, or sources derived therefrom, "isolated protein" (1) is unrelated to proteins found in nature, (2) is free of other proteins from the same source, e.g., is free of marine proteins, (3) is expressed by cells of a different species, or (4) does not occur in nature.

The term "polypeptide" is used herein as a generic term to refer to a native protein, fragment or analog of a polypeptide sequence. Thus, natural protein fragments and analogs are species of the genus Polypeptides.

As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) is naturally occurring and can be isolated from nature and not intentionally modified by man in the laboratory.

The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over a comparison window. The term "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, determining the number of matched positions by the occurrence of residues in either (e.g., a, T, C, G, U, or I) or both sequences, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence having at least 85% sequence identity, e.g., at least 90% to 95% sequence identity, with at least 99% sequence identity as compared to a reference sequence, typically over a comparison window of at least 18 nucleotide (6 amino acid) positions, typically over a window of at least 24-48 nucleotide (8-16 amino acid) positions, as compared to the reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may include deletions or additions that comprise 20% or less of the reference sequence over the entire comparison window. The reference sequence may be a subset of a larger sequence.

As used herein, twenty conventional amino acids and their abbreviations follow conventional usage see Immunology-ASynthesis (second edition, e.s. gold and d.r.gren, edited by Sinauer Associates, Sunderland7Mass. (1991)). twenty conventional amino acids, stereoisomers (e.g., D-amino acids) of unnatural amino acids (e.g., α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids) may also be polypeptides of the present disclosure examples of unconventional amino acids include 4-hydroxyproline, γ -carboxyglutamic acid, ε -N, N-trimethyllysine, ε -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ -N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).

Similarly, unless otherwise indicated, the left end of a single-stranded polynucleotide sequence is the 5 'end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5 'to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction sequence region on the DNA strand, which has the same sequence as the RNA and is located at the 5 'to 5' end of the RNA transcript as the "upstream sequence", and the sequence region on the DNA strand, which has the same sequence as the RNA and is at the 3 'to 3' end of the RNA transcript, is referred to as the "downstream sequence".

The term "substantial identity" as applied to polypeptides means that two peptide sequences have at least 80% sequence identity, e.g., at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity, when optimally aligned, e.g., using default GAP weights by the programs GAP or BESTFIT.

In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic hydroxyl side chains are serine and threonine. A group of amino acids having amide side chains is asparagine and glutamine; amino acid groups having aromatic side chains are phenylalanine, tyrosine and tryptophan. One group of amino acids with basic side chains is lysine, arginine and histidine. And a group of amino acids having sulfur-containing side chains are cysteine and methionine. Suitable conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic acid-aspartic acid and asparagine-glutamine.

As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated as being encompassed by the present disclosure provided that the changes in the amino acid sequence remain at least 75%, e.g., at least 80%, 90%, 95%, or 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur in families of amino acids that are related in side chain. Genetically encoded amino acids are generally classified into the following groups: (1) the acidic amino acid is aspartic acid or glutamic acid; (2) the basic amino acid is lysine, arginine, histidine; (3) the nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) the uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include: (i) serine and threonine, which belong to the aliphatic hydroxyl family; (ii) asparagine and glutamine, which belong to the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, which belong to the aliphatic family; (iv) phenylalanine, tryptophan and tyrosine, which belong to the aromatic family. For example, it is reasonably expected that a substitution of isoleucine or valine alone for leucine, glutamic for aspartic acids, serine for threonine, or a similar substitution of a structurally related amino acid for an amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within the framework site. Whether an amino acid change results in a functional peptide can be readily determined by determining the specific activity of the polypeptide derivative. The assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art. Suitable amino and carboxy termini of fragments or analogs occur near the boundaries of the functional domains. Domains and functional domains can be identified by comparing nucleotide and/or amino acid sequence data to public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al, Science 253: 164(1991). Thus, the foregoing examples demonstrate that one skilled in the art can identify sequence motifs and structural conformations that can be used to define the structures and functional domains according to the present invention.

The following amino acid substitutions are suitable: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for protein complex formation, (4) altered binding affinity, (4) conferring or modifying the physicochemical or functional properties of other such analogs. Analogs can include various muteins of a sequence different from that of the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside of the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to disrupt the helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). In Proteins, Structures and molecular principles (creative, editors, w.h. freeman and Company, New York (1984)), innovative Protein Structures (c.branden and j.toze, incorporated by Garland Publishing, New York, n.y. (1991)), and Thornton et al, Nature 354: 105(1991) examples of art-recognized secondary and tertiary structures of polypeptides are described.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion, but wherein the remaining amino acid sequence is identical at the corresponding position to the native sequence from which it was derived (e.g., from a full-length cDNA sequence). Fragments are typically at least 5, 6, 8 or 10 amino acids long, for example at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or at least 70 amino acids long. The term "analog" as used herein refers to a polypeptide consisting of a fragment of at least 25 amino acids that has substantial identity to a portion of the amino acid sequence from which it is derived and that has specific binding to CD47 under suitable binding conditions. Typically, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) relative to the native sequence. Analogs are typically at least 20 amino acids long, e.g., at least 50 amino acids long or longer, and often can be as long as a full-length naturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptidomimetics" or "peptidomimetic" (peptidomimetics). Fauchere, j.adv.drug res.15: 29(1986), Veber and Freidinger TINSp.392 (1985); and Evans et al, j.med.chem.30: 1229(1987). Such compounds are typically developed with the aid of computer molecular models. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are similar in structure to exemplary polypeptides (i.e., polypeptides having biochemical or pharmacological activity), such as human antibodies, but have one or more peptide bonds optionally substituted by a bond selected from the group consisting of: - - -CH₂NH--、--CH₂S-、--CH₂-CH₂-, - -CH- - - - (cis and trans) - -, - -COCH₂--、CH(OH)CH₂- - -and- -CH₂SO- -. Systematic substitution of one or more amino acids of the consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to produce more stable peptides. Additionally, constrained peptides comprising a consensus sequence or substantially the same consensus sequence variations can be generated by methods known in the art (Rizo and GieraschAnn. Rev. biochem. 61: 387(1992)), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bonds that cyclize the peptide.

The term "agent" is used herein to refer to a compound, a mixture of compounds, a biological macromolecule, and/or an extract made from a biological material.

As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, e.g., by incorporation of a radiolabeled amino acid or polypeptide attached to a biotin moiety, which may be labeled with avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by light or calorimetry.) In some cases, labels or tags may also be therapeutic.A variety of methods of labeling polypeptides and glycoproteins are known In the art and may be used.examples of polypeptide labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide series), enzymatic labels (e.g., horseradish peroxidase, β -luciferase, alkaline phosphatase), chemiluminescence, galactosyl, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, tags).

The term "antineoplastic agent" as used herein refers to an agent having functional properties that inhibit the development or progression of a human tumor, particularly a malignant (cancerous) lesion such as a carcinoma, sarcoma, lymphoma or leukemia. Metastasis inhibition is often a property of antineoplastic agents.

As used herein, the terms "treat," "treated," "treatment regimen," and the like refer to the alleviation and/or amelioration of the disorders and/or symptoms associated therewith. "alleviate" and/or "reduced" refers to reducing, inhibiting, attenuating, reducing, arresting and/or stabilizing the development or progression of a disease, such as cancer. It is to be understood that, although not excluded, treating a disease or condition does not require complete elimination of the disease, condition or symptom associated therewith.

Other Chemical Terms herein are used according to conventional usage in The art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. eds., McGraw-Hill, San Francisco (1985)).

As used herein, "substantially pure" means that the target species is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis), and in some embodiments, the substantially purified fraction is at least about 50% (on a molar basis) of all macromolecular species present in the composition that the target species is.

Generally, a substantially pure composition will comprise greater than about 80%, e.g., greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In some embodiments, the target substance is purified to substantial homogeneity (contaminants cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

In the present disclosure, "comprise," "include," "have," and the like may have meanings assigned to them by U.S. and/or european patent laws, and may mean "include," "include," and "contain," and the like; the term "consisting essentially of has the meaning as given in the united states patent law and these terms are open-ended, allowing more than the recited content, provided that the content does not change its basic or novel characteristics beyond the recited content, but excludes embodiments of the prior art.

An "effective amount" refers to the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound for practicing the present invention to treat a disease depends on the mode of administration, age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an "effective amount".

By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, rodent, sheep, primate, camelid, or feline.

As used herein, the term "administering" refers to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

Drawings

The following is a brief summary of the drawings.

FIG. 1: panitumumab anti-EGFR binders (black) and humanized SP34 anti-CD 3 binders (grey) assembled into various different bed structures.

FIG. 2: flow cytometric analysis of 3a6 and 10E6 hybridoma candidates on BAF cells expressing membrane bound EGFR. The figure shows FACS spectra of parental 3a6 and 10E6 hybridoma supernatants for membrane-bound EGFR expressed on BAF cells. 100 μ l harvested from two hybridoma clones were incubated with 100ul of EGFR-transfected BAF cells diluted at 106 cells/ml. As a negative control, purified mouse IgG isotype diluted at 10 μ g/ml was used. Antibody binding was detected with goat anti-mouse IgG-PE.

FIG. 3: 3A6a12B5 and 10E6F5 specifically bind to the extracellular domain IV of the EGFR receptor. The figure shows ELISA results in which the IV domain (D) of EGFR was subcloned against immobilized recombinant soluble EGFR (a) or EGFR-Her3 chimeric molecules (B and C) or single purified 3A6a12F5 and 10E6F5 hybridoma cells at several concentrations (10 to 0.01 μ g/ml).

Tests were also performed in the analysis.

FIG. 4: CD3-EGFR _5 and CD3-EGFR _8 showed killing activity against EGFR + A549 target cells. EGFR + a549 cells (target cells, T) were subjected to CD3 redirected killing assay using PBMC from 3 healthy donors as effector cells (E) at an E: T ratio of 10: 1 within 48 hours. The histogram shows the average percentage of specific killing calculated from 3 individual donors. Two BEAT molecules were used at 10nM in the assay.

FIG. 5: (A) KD measurement of chimeric 3a6 antibody. (B) KD measurement of chimeric 10E6 antibody.

FIG. 6: (A) KD measurement of 10E6 optimal antibody. (B) KD measurement of 10E6 stable antibody.

FIG. 7: (A) sensorgram for binding assay with 3a6 chimeric antibody, and sensorgram for binding assay with 10E6 chimeric antibody. (C) Sensorgram of control experiment using polyclonal goat anti-EGFR antibody.

FIG. 8: (A) thermogram of the optimal antibody of 10E 6. The first peak corresponds to the IgG1 CH2-CH3 domain, showing a Tm of 71.7 ℃ and the second peak corresponds to the Fab. (B)10E6 thermogram of stabilized antibody. The first peak corresponds to the IgG1 CH2-CH3 domain, showing a Tm of 71.8 ℃ and the second peak corresponds to the Fab.

FIG. 9: CD3xEGFR _1 was not effective in a549 tumors.

FIG. 10: CD3xEGFR-SF1 and CD3xEGFR-SF3 have the same efficacy in a549 tumors.

FIG. 11: CD3xEGFR-SF3 showed better efficacy than victibix (Vectibix) in SNU-216 tumors.

FIG. 12: effect of dexamethasone on the antitumor activity of CD3xEGFR-SF3 in the xenograft model. (A) The figure shows the mean tumor size (mm)³) + -SEM. (B) The figure shows tumor growth of each mouse at day 37.

FIG. 13: schematic representation of simple binding ELISA formats for EGFR (A) and CD3 (B).

FIG. 14: schematic representation of the dual binding ELISA format.

FIG. 15: CD3xEGFR-SF3 was detected in mouse sera by simple EGFR binding ELISA.

FIG. 16: CD3xEGFR-SF3 was detected in mouse serum by simple CD3 binding ELISA.

FIG. 17: CD3xEGFR-SF3 was detected in mouse serum by a dual CD3 and EGFR binding ELISA.

FIG. 18: pharmacokinetic profile of CD3xEGFR-SF3 in serum from Sprague-Dawley rats. The pharmacokinetics of CD3xEGFR-SF3 were evaluated in male Sprague-Dawley rats (n-4) after a single intravenous injection at 1mg/kg body weight. Blood samples (six weeks) for Pharmacokinetic (PK) assessments were collected at predetermined time points of 0.25, 1, 6, 24, 48, 96, 168, 336, 530, 672, 840 and 1008 hours at predetermined times of 42 days post-dose. The CD3xEGFR-SF3 concentration in these serum samples was quantified using a suitable ELISA method. Data representing four animals tested (N ═ 1).

FIG. 19: CD3xEGFR-SF3 binding was detected by ELISA. Dose responses of CD3xEGFR-SF3 and control antibodies were incubated on coated human CD3-Fc (huCD3-Fc, A), human EGFR domain I-IV histidine tag (huEGFR-His; B), or huEGFR-His (C), followed by coupling with anti-human IgG Fab to HRP (A and B) or huCD 3-biotin, followed by detection with HRP-coupled streptavidin (C). The graph shows sigmoidal dose-response binding curves (absorbance at 450 nM) for each treatment. Each data point is the mean ± SEM of replicate values from three independent replicates.

FIG. 20: CD3xEGFR-SF3 binding was detected by flow cytometry. The dose response of CD3xEGFR-SF3 and control antibody was incubated on PBMCS (A-C) or squamous carcinoma cell line NCI-H1703(D) and detected with PE-labeled anti-human IgG (Fc- γ). For PBMCs, cells were also labeled with anti-CD 4 or anti-CD 8 antibodies. These figures show the non-linear sigmoidal regression binding curves for the Mean Fluorescence Intensity (MFI) of each treatment. Each data point is the mean ± SEM of replicate values from three independent replicates.

FIG. 21: CD3xEGFR-SF3 induces targeted lysis of EGFR expressing human cancer cell lines. Target cancer cells (T) and effector cells (E; PBMC) were incubated at an E: T ratio of 1: 10 in the presence of dose response of CD3xEGFR-SF3 or a control antibody, and redirected lysis of cancer cells was determined by cytotoxicity assays (MTS). Extraction of EC from sigmoidal dose-response curves for specific killing₅₀The value is obtained. Error bars represent mean ± SEM. The cell lines redirected for lysis were statistically different (one-way anova; F ═ 5, 6; p < 0.0001).

FIG. 22: CD3xEGFR-SF3 has the potential for low antibody-dependent cell-mediated cytotoxicity. Antibody-dependent cell-mediated cytotoxicity (ADCC) of CD3xEGFR was evaluated in EGFR + cancer cell lines A-431 and A549(A) and CD3+ HPB-ALL cells (B) and using sigmoidal dose response curves from specific kills. Error bars represent mean ± SEM from two independent experiments. The therapeutic effect was statistically significant for EGFR + cancer cells (least squares model, F29, p < 0.0001) and CD3+ HPB-ALL cells (T test, T3, p < 0.05). Statistically significant differences (p < 0.05) are indicated by asterisks.

FIG. 23: CD3xEGFR-SF3 has no complement dependent cytotoxicity. Specific Complement Dependent Cytotoxicity (CDC) was assessed in EGFR + cancer cells a549(a) and CD3+ HPB-ALL cells (B) and sigmoidal dose response curves for specific CDCs are shown.

FIG. 24: effect of CD3xEGFR-SF3 on PBMC proliferation. PBMCs were incubated for 48 hours in the presence of increasing doses of CD3xEGFR or control. The figure shows the results of 3H-thymidine incorporation from six independent experiments. AE042, P1069 and TRS represent different batches of CD3xEGFR-SF3 at concentrations of 0.0005, 0.005, 0.05, 0.5 and 5 (in ug/ml), respectively. For the different treatments, "c" represents coating and "s" soluble. Error bars represent mean ± SEM.

Figure 25 statistical analysis of CD3xEGFR-SF3 effect on PBMC proliferation data in figure 24 were analyzed by fitting a least squares model, followed by Dunnett comparisons (α ═ 0,05) to compare the mean values of the no mAb control (a) and the isotype control (B) significant differences in the mean values are shown as lines of needles that are beyond decision limits (95% CI interval for each treatment; grey areas).

FIG. 26: non-specific CD4+ T cell activation in response to CD3xEGFR-SF 3. PBMCs were incubated for 24h or 48h in the presence of increasing doses of CD3xEGFR or control. Activation of CD4+ T cells was measured by flow cytometry as expression of activation marker CD 69. AE042, P1069 and TRS represent different batches of CD3xEGFR-SF3 at concentrations of 0.0005, 0.005, 0.05, 0.5 and 5ug/ml, respectively. For the different treatments, "c" represents coating and "s" soluble. Error bars represent mean ± SEM from six independent experiments.

Figure 27 statistical comparison of non-specific CD4+ T cell activation between CD3xEGFR-SF3 and no mAb condition the data in figure 26 was analyzed by fitting a least squares model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean to the 24 hour (a) and 48 hour (B) no mAb control.

Figure 28 statistical comparison of nonspecific CD4+ T cell activation between CD3xEGFR-SF3 and isotype control the data in figure 26 were analyzed using a fitted least squares model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean of 24h (a) and 48h (b) to isotype control.

FIG. 29: non-specific CD8+ T cell activation in response to CD3xEGFR-SF 3. PBMCs were incubated for 24h or 48h in the presence of increasing doses of CD3xEGFR-SF3 or control. Activation of CD8+ T cells was measured by flow cytometry as expression of activation marker CD 69. AE042, P1069 and TRS represent different batches of CD3xEGFR-SF3 at concentrations of 0.0005, 0.005, 0.05, 0.5 and 5ug/ml, respectively. For the different treatments, "c" represents coating and "s" soluble. Error bars represent mean ± SEM from six independent experiments.

Figure 30 statistical comparison of nonspecific CD8+ T cell activation between CD3xEGFR-SF3 and mAb-free conditions the data in figure 29 were analyzed using a least squares fit model, followed by a Dunnett comparison (α ═ 0.05) to compare the mean of 24h (a) and 48h (b) to the mAb-free control.

Figure 31 statistical comparison of nonspecific CD8+ T cell activation between CD3xEGFR-SF3 and isotype control the data in figure 29 were analyzed using a least squares fit model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean of 24h (a) and 48h (b) to isotype control.

Figure 32 h non-specific T cell cytokine response to CD3xEGFR-SF3 PBMCs were incubated for 24 hours in the presence of increasing doses of CD3xEGFR-SF3 or control and levels of released IL-2, IL-6, TNF- α and IFN- γ were measured in the supernatant by Luminex AE042 and P1069 represent different batches of CD3xEGFR-SF3 at concentrations of 0.0005, 0.005, 0.05, 0.5 and 5ug/ml respectively for different treatments, "c" represents coating, "s" represents soluble error bars represent mean ± SEM from six independent experiments.

Figure 33 statistical comparison of non-specific T cell cytokine responses between CD3xEGFR-SF3 and mAb-free conditions at 24h the data in figure 32 were analyzed by fitting a least squares model followed by a Dunnett comparison (a 0,05) to compare the mean with mAb-free controls of IL-2(a), IL-6(B), IFN- γ (C) and TNF- α (D) — significant differences in the mean are shown as lines of needles that exceed the decision limit (95% CI interval for each treatment; grey areas).

Figure 34 statistical comparison of nonspecific T cell cytokine responses between CD3xEGFR-SF3 and isotype control at 24h the data in figure 32 were analyzed by fitting a least squares model followed by a Dunnett comparison (a ═ 0,05) to compare the mean to isotype controls for IL-2(a), IL-6(B), IFN- γ (C) and TNF- α (D). significant differences in the mean are shown as lines of needles that exceed the decision limit (95% CI interval for each treatment; grey areas).

Figure 35 non-specific T cell cytokine response to CD3xEGFR-SF3 at 48h PBMC were incubated for 48 hours in the presence of increasing doses of CD3xEGFR-SF3 or control and levels of released IL-2, IL-6, TNF- α and IFN- γ were measured in the supernatant by Luminex AE042, P1069 and TRS for different batches of CD3xEGFR-SF3 at concentrations of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10ug/ml, respectively, "c" for coating, "s" for soluble. error bars represent mean ± SEM from six independent experiments.

Figure 36 statistical comparison of non-specific T cell cytokine responses between CD3xEGFR-SF3 and mAb-free conditions at 48h the data in figure 35 were analyzed by fitting a least squares model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean to mAb-free controls of IL-2(a), IL-6(B), IFN- γ (C) and TNF- α (D) — significant differences in the mean are shown as lines of dots that exceed the decision limit (95% CI interval for each treatment; grey areas).

Figure 37 statistical comparison of nonspecific T cell cytokine responses between CD3xEGFR-SF3 and isotype control at 48h the data of figure 35 were analyzed by fitting a least squares model followed by a Dunnett comparison (α ═ 0,05) to compare the mean to isotype controls for IL-2(a), IL-6(B), IFN- γ (C) and TNF- α (D). significant differences in the mean are shown as lines of dots that exceed the decision limit (95% CI interval for each treatment; grey areas).

FIG. 38: CD3xEGFR-SF3 did not induce a non-specific T cell cytokine response in the high density PBMC assay. PBMC were grown at high density (10)⁷Cells/m 1) for 48 hours. The cells were then grown at normal density (10)⁶Cells/ml) were plated and cultured for 24 hours in the presence of increasing doses of CD3xEGFR-SF3 or control and levels of IL-2, IL-6, TNF- α and the release of IFN- γ in the supernatant was measured by Luminex AE042 and TRS represent different batches of CD3xEGFR-SF3 concentrated in itThe degrees were 0.0001, 0.001, 0.01, 0.1, 1 and 10ug/ml, respectively. Error bars represent mean ± SEM from four independent experiments.

Figure 39 statistical comparison of non-specific T cell cytokine responses between CD3xEGFR-SF3 and no mAb condition in high density PBMC analysis the data in figure 38 were analyzed with a fitted least squares model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean to no mAb control of IL-2(a), IL-6(B), IFN- γ (C) and TNF-a (d) — significant differences in the mean are shown as lines of needles that exceed the decision limit (95% CI interval for each treatment; grey areas).

Figure 40 statistical comparison of nonspecific T cell cytokine responses between CD3xEGFR-SF3 and isotype control in high density PBMC analysis the data of figure 38 were analyzed by fitting a least squares model, followed by a Dunnett comparison (α ═ 0,05) to compare the mean to isotype controls for IL-2(a), IL-6(B), IFN- γ (C), and TNF- α (D) — significant differences in the mean are shown as lines of needles that exceed decision limits (95% CI interval for each treatment; grey areas).

FIG. 41 CD3xEGFR-SF3 did not induce cytokine responses in whole blood assays Whole blood from healthy volunteers was cultured for 24 hours in the presence of increasing doses of CD3xEGFR-SF3 or controls and the levels of IL-2, IL-6, TNF- α, and IFN- γ were measured in serum by Luminex AE042 and TRS represent different batches of CD3xEGFR-SF3 at concentrations of 0.001, 0.01, 0.1, and 1ug/ml respectively.

Figure 42 statistical comparison of cytokine responses between CD3xEGFR-SF3 and mAb-free conditions in whole blood assays the data in figure 41 were analyzed by fitting a least squares model, followed by Dunnett comparisons (a ═ 0,05) to compare the mean to mAb-free controls for IL-2(a), IL-6(B), IFN- γ (C), and TNF- α (D).

Figure 43 statistical comparison of cytokine responses between CD3xEGFR-SF3 and isotype control in whole blood assays the data of figure 41 were analyzed by fitting a least squares model, followed by Dunnett comparisons (α ═ 0,05) to compare the mean values to isotype controls for IL-2(a), IL-6(B), IFN- γ (C) and TNF- α (D) — significant differences in the mean values are shown as lines of stitches beyond the decision limit (95% CI interval for each treatment; grey areas).

FIG. 44: efficacy of CD3xEGFR-SF3 therapeutic treatment in NOD SCID xenograft mouse model. Expression levels of EGFR on a549 cells were determined by sABC before the figure. A mixture of tumor cells (target cells, T) and PBMCs (effector cells, E) was injected subcutaneously. The right abdominal region of nod.cb17/AlhnRj-Prkdcscid/Rj (NOD/SCID) mice (n ═ 4 to 5 per PBMC donor per group) was injected at an E: T ratio of 2: 1. CD3xEGFR-SF3 was administered intravenously. Starting on day 2, 2mg/kg was administered once a week for 3 weeks. Tumor growth was determined by external caliper measurements. These figures show the mean tumor size (mm)³) + -SEM. 2 PBMC donors were included. Study name: a549_ 15.

FIG. 45: on day 41, the A549 tumor volumes of CD3xEGFR-SF3 were compared between the treated and control groups. The data show the tumor volume of each animal on day 41 for each group. Data is extracted from fig. 44. Study name: a549J _ 5.

Detailed Description

The following provides a set of non-exhaustive examples relating to the present invention.