HK1185085B

HK1185085B - Human monoclonal antibodies to fucosyl-gm1 and methods for using anti-fuco-syl-gm1

Info

Publication number: HK1185085B
Application number: HK13112220.2A
Authority: HK
Inventors: C．A．维斯蒂卡; E．H．霍尔梅斯; P．布拉姆斯; A．威特; J．M．卡尔达勒利
Original assignee: E.R. Squibb & Sons, L.L.C.
Priority date: 2005-12-08
Filing date: 2013-10-30
Publication date: 2017-06-09

Description

Human monoclonal antibodies to fucosyl-GM 1 and methods of using anti-fucosyl-GM 1 antibodies

The present application is a divisional application of invention patent application No. 200680050725.5, entitled "human monoclonal antibody against fucosyl-GM 1 and method of using anti-fucosyl-GM 1 antibody" filed on 8.12.2006.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial No. 60/748,915, filed on 8/12/2005, which is incorporated herein by reference.

Background

fucosyl-GM 1 is a sphingolipid monosialoganglioside, which consists of a ceramide lipid component that anchors molecules in the cell membrane and a carbohydrate component that is exposed on the cell surface. Carbohydrate antigens are the most abundantly expressed antigens on the surface of cancer cells (Feizi T. (1985) Nature 314: 53-7). For certain tumor types, such as Small Cell Lung Cancer (SCLC), there is an initial effective response to chemotherapy, but a rapid onset of chemotherapy-refractory relapse follows. Intervention with novel immunotherapeutic drugs can successfully overcome drug resistance relapse (Johnson DH (1995) Lungcancer12Suppl3: s71-5). Several carbohydrate antigens, such as gangliosides GD3 and GD2, have been shown to function as effective targets for passive immunotherapy using MAbs (Irie RF and Morton DL (1986) PNAS 83: 8694-. Also in clinical trialsGanglioside antigens have been shown to be effective targets for active immunotherapy using vaccines (Krug LM et al (2004) Clinical Cancer Research 10: 6094-. Indeed, sera from SCLC patients who produce anti-fucosyl-GM 1 antibody titers after vaccination with KLH-conjugated antigens have been shown to bind specifically to tumor cells and have tumor-specific complement-dependent cytotoxicity (CDC). Toxicity associated with anti-fucosyl-GM 1 titers was mild and transient, with three limited-phase SCLC patients not relapsing at 18, 24, 30 months (Krug et al, supra; Dickler et al, supra).

fucosyl-GM 1 has been shown to be expressed in a high percentage of SCLC cases, unlike other ganglioside antigens, fucosyl-GM 1 is rarely or not expressed in normal tissues (Nilsson et al (1984) glycon junction J1: 43-9; Krug et al, supra; Brezicka et al (1989) Cancer Res 49: 1300-5; Zhanggyi et al (1997) Iht J Cancer 73: 42-49; Brezicka et al (2000) Lung Cancer 28: 29-36; Fredman et al (1986) Biochim Acta: 316-23; Brezicka et al (1991) AP 99: 797-802; Nilsson et al (1986) Cancer Res 46: 875). The presence of fucosyl-GM 1 has been demonstrated in the culture medium of SCLC cell lines, in tumor extracts and sera of xenografted nude mice, and in the sera of SCLC patients with stage disease (Vangsted et al (1991) Cancer Res 51: 2879-84; Vangsted et al (1994) Cancer Detect Prev 18: 221-9). These reports provide convincing evidence that fucosyl-GM 1 is a highly specific tumor antigen that can be targeted by immunotherapeutic agents.

Thus, there is a need for agents that recognize fucosyl-GM 1 and methods of using such agents.

Disclosure of Invention

The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that bind fucosyl-GM 1 and exhibit a number of desirable properties. These properties include high affinity binding to fucosyl-GM 1, and binding to human small cell lung cancer cell line DMS-79 (human SCLC ATCC # CRL-2049). Methods of using the antibodies and compositions of the invention to treat a variety of fucosyl-GM 1-mediated diseases are also provided.

In one aspect, the invention relates to an isolated monoclonal antibody, or antigen binding portion thereof, wherein the antibody (a) specifically binds to fucosyl-GM 1; and (b) binds to human small cell lung cancer cell line DMS-79 (human SCLC ATCC # CRL-2049).

Preferably, the antibody is a human antibody, but in alternative embodiments, the antibody may also be a murine antibody, a chimeric antibody, or a humanized antibody.

In another embodiment, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, wherein the antibody cross-competes for binding to fucosyl-GM 1 with a reference antibody, wherein the reference antibody (a) specifically binds to fucosyl-GM 1; and (b) binds to human small cell lung cancer cell line DMS-79 (human SCLC ATCC # CRL-2049). In certain embodiments, the reference antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 1; and (b) comprises SEQ ID NO: 7, or a light chain variable region of the amino acid sequence of seq id No. 7. In further embodiments, the reference antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 2; and (b) comprises SEQ ID NO: 8 in a light chain variable region of the amino acid sequence of seq id No. 8. In other embodiments, the reference antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 3; and (b) comprises SEQ ID NO: 9 in the amino acid sequence of seq id no. In further embodiments, the reference antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 4; and (b) comprises SEQ ID NO: 10, or a light chain variable region of the amino acid sequence of seq id No. 10. In further embodiments, the reference antibody comprises: (a) comprises SEQ ID NO: 5; and (b) comprises SEQ ID NO: 11, or a light chain variable region of the amino acid sequence of seq id No. 11. In further embodiments, the reference antibody comprises: (a) comprises the amino acid sequence of SEQ ID NO: 6; and (b) comprises SEQ ID NO: 12.

In another aspect, the invention relates to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a monoclonal antibody derived from human V_H3-48 gene or a heavy chain variable region which is the product of the gene, wherein the antibody specifically binds to fucosyl-GM 1. The invention further provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a monoclonal antibody derived from human V_KAn L15 gene or a light chain variable region which is the product of the gene, wherein the antibody specifically binds to fucosyl-GM 1.

A preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 13, heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 19 heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 25, heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 31, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 37, a light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 43 CDR3 of the light chain variable region.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 14, a heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 20, a heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 26, a heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 32, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 38, light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 44, CDR3 of the light chain variable region.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 15, heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 21, heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 33, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 39 light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 45 CDR3 of the light chain variable region.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 22, heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 28, heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 34, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 40 light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 46 light chain variable region CDR3.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 17, a heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 23, heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 29, heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 35, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 41 light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 47 light chain variable region CDR3.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 18, heavy chain variable region CDR 1;

(b) comprises the amino acid sequence of SEQ ID NO: 24, a heavy chain variable region CDR 2;

(c) comprises the amino acid sequence of SEQ ID NO: 30, a heavy chain variable region CDR 3;

(d) comprises the amino acid sequence of SEQ ID NO: 36, light chain variable region CDR 1;

(e) comprises the amino acid sequence of SEQ ID NO: 42, light chain variable region CDR 2; and

(f) comprises the amino acid sequence of SEQ ID NO: 48 CDR3 of the light chain variable region.

Other preferred antibodies or antigen-binding portions thereof of the invention include:

(a) comprises the amino acid sequence of SEQ ID NO: 1; and

(b) comprises the amino acid sequence of SEQ ID NO: 7, or a light chain variable region of the amino acid sequence of seq id No. 7.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 2; and

(b) comprises the amino acid sequence of SEQ ID NO: 8 in a light chain variable region of the amino acid sequence of seq id No. 8.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 3; and

(b) comprises the amino acid sequence of SEQ ID NO: 9 in the amino acid sequence of seq id no.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 4; and

(b) comprises the amino acid sequence of SEQ ID NO: 10, or a light chain variable region of the amino acid sequence of seq id No. 10.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 5; and

(b) comprises the amino acid sequence of SEQ ID NO: 11, or a light chain variable region of the amino acid sequence of seq id No. 11.

Another preferred combination comprises:

(a) comprises the amino acid sequence of SEQ ID NO: 6; and

(b) comprises the amino acid sequence of SEQ ID NO: 12.

The antibody of the invention may be, for example, a full length antibody such as an IgG1 or IgG4 isotype. Alternatively, the antibodies may be antibody fragments, such as Fab or Fab'₂Fragments or single chain antibodies.

The invention also provides an immunoconjugate comprising an antibody or antigen-binding portion thereof of the invention linked to a therapeutic agent, such as a cytotoxin or a radioisotope. The invention also provides a bispecific molecule comprising an antibody or antigen-binding portion thereof of the invention linked to a second functional portion having a different binding specificity than the antibody or antigen-binding portion thereof.

Also provided are compositions comprising an antibody or antigen-binding portion thereof or immunoconjugate or bispecific molecule of the invention and a pharmaceutically acceptable carrier.

The invention also includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of the invention, as well as expression vectors comprising these nucleic acids, host cells comprising these expression vectors. Furthermore, the invention provides a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses an antibody of the invention, as well as hybridomas made from such a mouse, wherein the hybridomas produce an antibody of the invention.

In another aspect, the invention provides a method of treating or preventing a disease characterized by growth of fucosyl-GM 1-expressing tumor cells, comprising administering to a subject an amount of an antibody, or antigen-binding portion thereof, of the invention effective to treat or prevent the disease. The disease may be, for example, cancer, such as lung cancer (including small cell lung cancer).

In a preferred embodiment, the present invention provides a method of treating cancer in vivo using an anti-fucosyl-GM 1 antibody. The anti-fucosyl-GM 1 antibody may be a murine, chimeric, humanized or human antibody. Examples of other cancers that may be treated by the methods of the invention include lung cancer (including small cell lung cancer and non-small cell lung cancer), kidney cancer (e.g., renal cell carcinoma), glioblastoma, brain tumors, chronic or acute leukemias (including Acute Lymphocytic Leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphomas (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, burkitt's lymphoma, Anaplastic Large Cell Lymphoma (ALCL), cutaneous T-cell lymphoma, nodular small-oolytic lymphoma, peripheral T-cell lymphoma, lunett lymphoma, immunoblastic lymphoma, T-cell leukemia/lymphoma (ATLL) Central blast/central cellular (cb/cc) follicular lymphoma, B-lineage diffuse large cell lymphoma, angioimmunoblastic lymphadenopathy (AILD) -like T-cell lymphoma and HIV-associated body cavity lymphoma), embryonal carcinoma, undifferentiated nasopharyngeal carcinoma (e.g., schmingkin's tumor), castleman's disease, kaposi's sarcoma, multiple myeloma, waldenstrom's macroglobulinemia and other B-cell lymphomas, nasopharyngeal carcinoma, bone cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, cervical cancer, Bladder cancer, kidney or ureter cancer, renal pelvis cancer, Central Nervous System (CNS) tumors, tumor angiogenesis, spinal column tumors, brain stem glioma, pituitary adenoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers, such as mesothelioma), and combinations of said cancers.

Other features and advantages of the present invention will be apparent from the following detailed description and examples, which are not to be construed in a limiting sense. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Drawings

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 49) and amino acid sequence (SEQ ID NO: 1) of the heavy chain variable region of the 5B1 human monoclonal antibody. The CDR1(SEQ ID NO: 13), CDR2(SEQ ID NO: 19) and CDR3(SEQ ID NO: 25) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 55) and amino acid sequence (SEQ ID NO: 7) of the variable region of the light chain of the 5B1 human monoclonal antibody. The CDR1(SEQ ID NO: 31), CDR2(SEQ ID NO: 37) and CDR3(SEQ ID NO: 43) regions are outlined, and the germline sources of V and J are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 50) and amino acid sequence (SEQ ID NO: 2) of the heavy chain variable region of 5B1a human monoclonal antibody. The CDR1(SEQ ID NO: 14), CDR2(SEQ ID NO: 20) and CDR3(SEQ ID NO: 26) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 56) and amino acid sequence (SEQ ID NO: 8) of the variable region of the light chain of the 5B1a human monoclonal antibody. The CDR1(SEQ ID NO: 32), CDR2(SEQ ID NO: 38) and CDR3(SEQ ID NO: 44) regions are outlined, and the germline sources of V and J are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 51) and amino acid sequence (SEQ ID NO: 3) of the heavy chain variable region of the 7D4 human monoclonal antibody. The CDR1(SEQ ID NO: 15), CDR2(SEQ ID NO: 21) and CDR3(SEQ ID NO: 27) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 57) and amino acid sequence (SEQ ID NO: 9) of the variable region of the light chain of the 7D4 human monoclonal antibody. The CDR1(SEQ ID NO: 33), CDR2(SEQ ID NO: 39) and CDR3(SEQ ID NO: 45) regions are outlined, and the germline sources of V and J are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 52) and amino acid sequence (SEQ ID NO: 4) of the heavy chain variable region of the 7E4 human monoclonal antibody. The CDR1(SEQ ID NO: 16), CDR2(SEQ ID NO: 22) and CDR3(SEQ ID NO: 28) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 58) and amino acid sequence (SEQ ID NO: 10) of the variable region of the light chain of the 7E4 human monoclonal antibody. The CDR1(SEQ ID NO: 34), CDR2(SEQ ID NO: 40) and CDR3(SEQ ID NO: 46) regions are outlined, and the germline sources of V and J are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 5) of the heavy chain variable region of the 13B8 human monoclonal antibody. The CDR1(SEQ ID NO: 17), CDR2(SEQ ID NO: 23) and CDR3(SEQ ID NO: 29) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 59) and amino acid sequence (SEQ ID NO: 11) of the variable region of the light chain of the 13B8 human monoclonal antibody. The CDR1(SEQ ID NO: 35), CDR2(SEQ ID NO: 41) and CDR3(SEQ ID NO: 47) regions are outlined, and the germline sources of V and J are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 54) and amino acid sequence (SEQ ID NO: 63) of the heavy chain variable region of the 3C4 human monoclonal antibody. The CDR1(SEQ ID NO: 65), CDR2(SEQ ID NO: 66) and CDR3(SEQ ID NO: 67) regions are outlined, and the germline sources of V, D and J are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 64) of the variable region of the light chain of the 3C4 human monoclonal antibody. The CDR1(SEQ ID NO: 68), CDR2(SEQ ID NO: 69) and CDR3(SEQ ID NO: 70) regions are outlined, and the germline sources of V and J are indicated.

FIG. 7 shows the amino acid sequences of the heavy chain variable regions of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 together with human germline V_H3-48 (SEQ ID NO: 61).

FIG. 8 shows the amino acid sequences of the light chain variable regions of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 together with human germline V_KAlignment of the L15 amino acid sequence (SEQ ID NO: 62).

FIGS. 9A to C show the results of ELISA experiments demonstrating that human monoclonal antibodies against fucosyl-GM 1 specifically bind to fucosyl-GM 1.

Fig. 10A to C show the results of whole cell ELISA experiments demonstrating that human monoclonal antibodies against fucosyl-GM 1 bind specifically to cells expressing fucosyl-GM 1.

Fig. 11A to C show the results of flow cytometry experiments demonstrating that (a and B) anti-fucosyl-GM 1 human monoclonal antibody binds to the cell surface of cell line DMS79, and (C) DMS79 cells were found to continue to express fucosyl-GM 1 in vivo (i.e. after implantation in mice).

FIGS. 12A and 12B show the results of Hum-Zap internalization experiments demonstrating that human monoclonal antibodies to fucosyl-GM 1 can be internalized to fucosyl-GM 1⁺Inside the cell.

FIGS. 13A and 13B show the results of a Complement Dependent Cytotoxicity (CDC) cell proliferation assay, demonstrating that the human monoclonal anti-fucosyl-GM 1 antibody kills the fucosyl-GM 1 expressing cell lines (A) DMS79 and (B) H-4-II-E.

Fig. 14A and 14B show the results of an antibody-dependent cellular cytotoxicity (ADCC) cell proliferation assay, demonstrating that human monoclonal anti-fucosyl-GM 1 antibody killed fucosyl-GM 1 expressing cell lines in the absence of CD16 blocking.

FIGS. 15A-C show implantation of DMS79 Small cell Lung cancer tumor cells (fucosyl-GM 1)⁺) (iii) tumor volume in individual SCID mice as a function of time. After tumor establishment, mice were treated five times with one of the following therapeutic agents: (A) PBS (vehicle control); (B) 30mg/kg human IgG1 per mouse (isotype control); (C) anti-fucosyl-GM 1 monoclonal antibody 5B1 at 10mg/kg per mouse; (D) anti-fucosyl-GM 1 monoclonal antibody 5B1 at 30mg/kg per mouse; (E) anti-fucosyl-GM 1 monoclonal antibody 7E4 at 10mg/kg per mouse; (F) anti-fucosyl-GM 1 monoclonal antibody 7E4 at 30mg/kg per mouse. The tumor volume on the first day of treatment was approximately 200mm³。

Fig. 16A and 16B show the mean and median, respectively, of tumor volumes for the mice shown in fig. 15.

FIG. 17 shows the mean body weights of groups of mice shown in FIG. 15.

Detailed Description

In one aspect, the invention relates generally to isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind fucosyl-GM 1. In certain embodiments, the antibodies of the invention exhibit one or more desired functional properties, such as high affinity binding to fucosyl-GM 1, and/or the ability to inhibit tumor cell growth in vitro or in vivo. In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain germline sequences, and/or comprise particular structural features, such as CDR regions comprising particular amino acid sequences. The invention provides isolated antibodies, methods of making the antibodies, immunoconjugates and bispecific molecules comprising the antibodies, and pharmaceutical compositions comprising the antibodies, immunoconjugates or bispecific molecules of the invention. The invention also relates to methods of using the antibodies, for example, to treat diseases such as cancer.

In order that the invention may be more readily understood, certain terms are first defined. Other definitions are set forth in the detailed description of the invention.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above-mentioned cells or the liver (including antibodies, cytokines, and complement) that results in the selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues.

"Signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of signals from one part of a cell to another. The phrase "cell surface receptor" as used herein includes, for example, molecules and molecular complexes that are capable of receiving a signal and propagating such a signal across the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is the fucosyl-GM 1 receptor.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. The heavy chain constant region is composed of three domains C_H1、C_H2And C_H3And (4) forming. Each light chain is composed of a light chain variable region (abbreviated herein as V)_L) And a light chain constant region. The light chain constant region consists of a domain C_LAnd (4) forming. V_HAnd V_LThe regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each V_HAnd V_LEach consisting of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens.The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., fucosyl-GM 1). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) fab fragments, i.e. consisting of V_L、V_H、C_LAnd C_H1Monovalent fragments consisting of domains; (ii) f (ab')₂Fragments, i.e. bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) from V_HAnd C_H1Domain-forming Fd fragments; (iv) v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) from V_HdAb fragments consisting of domains (Ward et al (1989) Nature 341: 544-546); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, despite the two domains V of the Fv fragment_LAnd V_HEncoded by separate genes, but they can be joined together by recombinant means by synthetic linkers that enable them to be made into a single protein chain, where V_LAnd V_HThe region pairs constitute monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained by conventional techniques well known to those skilled in the art and screened for utility in the same manner as intact antibodies.

As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds fucosyl-GM 1 is substantially free of antibodies that specifically bind antigens other than fucosyl-GM 1). Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "human antibody" as used herein includes antibodies having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant methods, for example: (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes for human immunoglobulin genes or hybridomas prepared therefrom (described further below), (b) antibodies isolated from transformed human antibody-expressing host cells, such as transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) by a process comprising splicing human immunoglobulin gene sequences to other DN' sAntibodies produced, expressed, produced or isolated by any other method for the A sequence. These recombinant human antibodies have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when transgenic animals using human Ig sequences, to in vivo endosomal cell mutagenesis), and thus the V of the recombinant antibody_HAnd V_LThe amino acid sequence of the region although derived from human germline V_HAnd V_LSequences and related sequences, but may not naturally occur in vivo in all components of the human antibody germline (properitoire).

The term "isotype" as used herein refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The phrases "antibody that recognizes an antigen" and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody and other agent or antibody.

The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Other framework region modifications may also be made within the human framework sequence.

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, for example, 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 "antibody specifically binding to fucosyl-GM 1" as used herein refers to the antibody expressed as 1 × 10^-7M or less, more preferably 5 × 10^-8M or less, more preferably 1 × 10^-8M or less, more preferably 5 × 10^-9K of M or less_DAn antibody that binds to fucosyl-GM 1.

The term "K" as used herein_assoc"or" K_a"refers to the binding rate of a particular antibody-antigen interaction, and the term" K "is used herein_dis"or" K_d"refers to the off-rate of a particular antibody-antigen interaction. The term "K" as used herein_D"refers to the dissociation constant, which is represented by K_dAnd K_aObtained by the ratio of (i.e. K)_d/K_a) And is expressed as molar concentration (M). K of antibody_DValues can be determined using methods established in the art. Determination of antibody K_DA preferred method of (2) is to use surface plasmon resonance, preferably using biosensor systems, e.g.Provided is a system.

The term "high affinity" of an IgG antibody as used herein refers to the K of the antibody for the target antigen_DIs 10^-8M or less, more preferably 10^-9M or less, even more preferably 10^-10M or less. However, for other antibody isotypes, "high affinity" binding may differ. For example, for an IgM isotype, "high affinity" binding means that the antibody has 10^-7M or less, more preferably 10^-8M or less, even more preferably 10^-9K of M or less_D。

The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.

Various aspects of the present application are described in further detail in the following sections.

anti-fucosyl-GM 1 antibody

The antibodies of the invention are characterized by specific functional features or characteristics of the antibodies. For example, these antibodies are specific for fucosyl-GM 1And (4) combining. Preferably, the antibodies of the invention bind fucosyl-GM 1 with high affinity, e.g., K_DIs 1 × 10^-7M or less. The anti-fucosyl-GM 1 antibody of the invention preferably exhibits one or more of the following characteristics:

(a) specifically bind to fucosyl-GM 1; and

(b) binds to human small cell lung cancer cell line DMS-79 (human SCLC ATCC # CRL-2049).

Preferably, the antibody is at 5 × 10^-8K of M or less_DCombined with fucosyl-GM 1 at 1 × 10^-8K of M or less_DCombined with fucosyl-GM 1 at 5 × 10^-9K of M or less_DCombined with fucosyl-GM 1 to be between 1 × 10^-8M and 1 × 10^-9K between M or lower_DBinding to fucosyl-GM 1. Standard assays for assessing the binding ability of antibodies to fucosyl-GM 1 are well known in the art and include, for example, ELISA, Western blot analysis and RIA. Binding kinetics (e.g., binding affinity) of an antibody can also be assessed by standard assays known in the art, such as ELISA, Scatchard and Biacore analysis.

Monoclonal antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5

Preferred antibodies of the invention include human monoclonal antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 isolated and structurally characterized as described in examples 1 and 2. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_HThe amino acid sequences are shown in SEQ ID NOs: 1.2, 3,4, 5 and 6. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_LThe amino acid sequences are shown in SEQ ID NOs: 7. 8,9, 10, 11 and 12.

Since each of these antibodies was able to bind to fucosyl-GM 1, these V s_HAnd V_LThe sequences may be "mixed and matched" to produce other anti-fucosyl-GM 1 binding molecules of the invention. The binding of fucosyl-GM 1 to these "mixed and matched" antibodies can be performed as described above and inDetection is by a binding assay (e.g., ELISA) as described in the examples. Preferably, when V_HAnd V_LWhen the chains are mixed and matched, from a particular V_H/V_LPaired V_HSequence is replaced by a structurally similar V_HAnd (4) sequencing. Also, preferably, from a particular V_H/V_LPaired V_LSequence is replaced by a structurally similar V_LAnd (4) sequencing.

Accordingly, in one aspect, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising:

(a) comprises a sequence selected from SEQ ID NO: 1.2, 3,4, 5 and 6; and

(b) comprises a sequence selected from SEQ ID NO: 7. 8,9, 10, 11 and 12;

wherein the antibody specifically binds to fucosyl-GM 1, preferably fucosyl-GM 1.

Preferred heavy and light chain combinations include:

(a) comprises the amino acid sequence of SEQ ID NO: 1; and (b) comprises SEQ ID NO: 7; or

(a) Comprises the amino acid sequence of SEQ ID NO: 2; and (b) comprises SEQ ID NO: 8; or

(a) Comprises the amino acid sequence of SEQ ID NO: 3; and (b) comprises SEQ ID NO: 9, a light chain variable region of the amino acid sequence of seq id no; or

(a) Comprises the amino acid sequence of SEQ ID NO: 4; and (b) comprises SEQ ID NO: 10, a light chain variable region of the amino acid sequence of seq id no; or

(a) Comprises the amino acid sequence of SEQ ID NO: 5; and (b) comprises SEQ ID NO: 11, or a light chain variable region of the amino acid sequence of seq id no; or

(a) Comprises the amino acid sequence of SEQ ID NO: 6; and (b) comprises SEQ ID NO: 12.

In another aspect, the invention provides antibodies comprising the heavy and light chain CDRs 1, CDR2, and CDR3 of 5B1, 5B1a, 7D4, 7E4, 13B8, and 18D5, or combinations thereof. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_HThe amino acid sequences of CDR1 are shown in SEQ ID NOs: 13. 14, 15, 16, 17 and 18. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_HThe amino acid sequences of CDR2 are shown in SEQ ID NOs: 19. 20, 21, 22, 23 and 24. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_HThe amino acid sequences of CDR3 are shown in SEQ ID NOs: 25. 26, 27, 28, 29 and 30. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_KThe amino acid sequences of CDR1 are shown in SEQ ID NOs: 31. 32, 33, 34, 35 and 36. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_KThe amino acid sequences of CDR2 are shown in SEQ ID NOs: 37. 38, 39, 40, 41 and 42. V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5_KThe amino acid sequences of CDR3 are shown in SEQ ID NOs: 43. 44, 45, 46, 47 and 48. CDR regions are delineated by the Kabat system (Kabat, E.A. et al (1991) Sequences of Proteins of immunological Interest, fifth edition, U.S. department of Health and Human Services, NIH publication No. 91-3242).

Since these antibodies all bind to fucosyl-GM 1 and antigen binding specificity is provided primarily by the CDR1, CDR2 and CDR3 regions, V_HCDR1, CDR2 and CDR3 sequences and V_KThe CDR1, CDR2, and CDR3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain a V_HCDR1, CDR2 and CDR3 and V_KCDR1, CDR2 and CDR3) to produce other anti-fucosyl-GM 1 binding molecules of the invention. The binding of fucosyl-GM 1 to these "mixed and matched" antibodies can be detected using the binding assays described above and in the examples (e.g., ELISA, Biacore analysis). Preferably, when V_HWhen CDR sequences are mixed and matched, from a particular V_HCDRs of sequences1. The CDR2 and/or CDR3 sequences are replaced with structurally similar CDR sequences. Likewise, when V_KWhen CDR sequences are mixed and matched, from a particular V_KThe CDR1, CDR2, and/or CDR3 sequences of the sequence are preferably replaced with structurally similar CDR sequences. As will be apparent to those skilled in the art, by combining one or more V_HAnd/or V_LReplacement of CDR region sequences with structurally similar sequences from the CDR sequences of monoclonal antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 disclosed herein can generate novel V_HAnd V_LAnd (4) sequencing.

Accordingly, in another aspect, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising:

(a) comprises a sequence selected from SEQ ID NO: 13. 14, 15, 16, 17 and 18, CDR 1;

(b) comprises a sequence selected from SEQ ID NO: 19. 20, 21, 22, 23 and 24, CDR2 of the heavy chain variable region of the amino acid sequence;

(c) comprises a sequence selected from SEQ ID NO: 25. 26, 27, 28, 29 and 30, CDR3 of the heavy chain variable region of the amino acid sequence;

(d) comprises a sequence selected from SEQ ID NO: 31. 32, 33, 34, 35 and 36, and a light chain variable region CDR 1;

(e) comprises a sequence selected from SEQ ID NO: 37. 38, 39, 40, 41 and 42, or a light chain variable region CDR 2; and

(f) comprises a sequence selected from SEQ ID NO: 43. 44, 45, 46, 47 and 48, or a light chain variable region CDR 3;

In a preferred embodiment, the antibody comprises:

In another preferred embodiment, the antibody comprises:

(c) comprises SEQ ID NO: 27, a heavy chain variable region CDR 3;

In another preferred embodiment, the antibody comprises:

It is well known in the art that independent of the CDR1 and/or CDR2 domains, the individual CDR3 domains can determine the binding specificity of an antibody to a homologous antigen, and that multiple antibodies with the same binding specificity can be predictively generated based on the common CDR3 sequence see, e.g., Klimka et al, British J.of Cancer 83 (2): 252-_vβ₃Set of humanized anti-integrin α of heavy and light chain variable CDR3 domains of antibody LM609_vβ₃An antibody, wherein each member antibody contains a different sequence outside of the CDR3 domain and is capable of binding the same epitope as a parent murine antibody with as high or higher affinity as the parent murine antibody); barbas et al, j.am.chem.soc.116: 2161-; barbas et al, Proc, natl.acad.sci.u.s.a.92: 2529 2533(1995) (grafting of the heavy chain CDR3 sequences of three Fab (SI-1, SI-40 and SI-32) against human placental DNA onto the heavy chain of the anti-tetanus toxoid Fab was described, thereby replacing the heavy chain CDR3 present and demonstrating that the CDR3 alone provides binding specificity); and Ditzel et al, j.immunol.157: 739 (1996) (describes a grafting study in which the transfer of the heavy chain CDR3 of the parent multispecific Fab LNA3 to only the heavy chain of a monospecific IgG tetanus toxoid conjugated Fab p313 antibody was sufficient to retain the binding specificity of the parent Fab). Each of the above references is incorporated by reference in its entirety.

Accordingly, in certain aspects, the invention provides a monoclonal antibody comprising one or more heavy and/or light chain CDR3 domains from a non-human antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to fucosyl-GM 1. In certain embodiments, these antibodies comprising one or more heavy and/or light chain CDR3 domains from a non-human antibody are capable of competing for binding with a corresponding parent non-human antibody (a); (b) functional characteristics are reserved; (c) binds to the same epitope; and/or (d) have similar binding affinities.

In other aspects, the invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from a first human antibody (e.g., a human antibody obtained from a non-human animal), wherein the first human antibody is capable of specifically binding to fucosyl-GM 1, and wherein a CDR3 domain from the first human antibody replaces the CDR3 domain in the human antibody lacking binding specificity for fucosyl-GM 1, thereby producing a second human antibody capable of specifically binding to fucosyl-GM 1. In certain embodiments, an antibody of the invention comprising one or more heavy and/or light chain CDR3 domains from a first human antibody is capable of competing for binding with a corresponding parent first human antibody (a); (b) functional characteristics are reserved; (c) binds to the same epitope; and/or (d) have similar binding affinities.

Antibodies with specific germline sequences

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the invention relates to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a monoclonal antibody derived from human V_H3-48 gene or a heavy chain variable region which is the product of the gene, wherein the antibody specifically binds to fucosyl-GM 1, preferably fucosyl-GM 1. In another preferred embodiment, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a monoclonal antibody derived from human V_KAn L15 gene or a light chain variable region which is the product of the gene, wherein the antibody specifically binds to fucosyl-GM 1, preferably fucosyl-GM 1. In another preferred embodiment, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereofWherein the antibody:

(a) comprising a protein of human origin V_H3-48 gene (the gene encodes the amino acid sequence shown in SEQ ID NO: 61) or the heavy chain variable region as the product of the gene;

(b) comprising a protein of human origin V_KThe L15 gene (the gene encodes the amino acid sequence shown in SEQ ID NO: 62) or the light chain variable region which is the product of the gene; and is

(c) Specifically binds to fucosyl-GM 1.

Respectively have V_H3-48 and V_kV of L15_HAnd V_KExamples of the antibody of (3) are 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D 5.

Herein, if the variable region of a human antibody is obtained from a system using human germline immunoglobulin genes, the human antibody comprises heavy or light chain variable regions "derived from" or "as a product of" a particular germline sequence. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest, or screening libraries of human immunoglobulin genes displayed on phage with the antigen of interest. Human antibodies "derived from" human germline immunoglobulin sequences or "as their products" can be identified by: the amino acid sequence of the human antibody is compared to the amino acid sequence of a human germline immunoglobulin and the human germline immunoglobulin sequence closest in sequence (i.e., having the highest% identity) to the human antibody sequence is selected. A human antibody "derived from" or "as a product of" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example due to deliberate introduction of naturally-occurring somatic mutations or site-directed mutations. However, a selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that, when compared to germline immunoglobulin amino acid sequences from other species (e.g., murine germline sequences), confirm that the human antibody is a human antibody. In certain instances, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from a particular human germline sequence exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain instances, the human antibody may exhibit no more than 5, or even no more than 4,3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

Homologous antibodies

In another embodiment, the antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibody retains the desired functional properties of the anti-fucosyl-GM 1 antibody of the invention.

For example, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises a heavy chain variable region and a heavy chain variable region selected from SEQ ID NOs: 1.2, 3,4, 5 and 6, which are at least 80% homologous in amino acid sequence;

(b) the light chain variable region comprises a heavy chain variable region substantially identical to a light chain variable region selected from the group consisting of SEQ ID NO: 7. 8,9, 10, 11 and 12, amino acid sequences that are at least 80% homologous; and is

The antibody exhibits one or more of the following properties:

(c) the antibody is expressed as 1 × 10^-7K of M or less_DBinding to fucosyl-GM 1;

(d) the antibody binds to human small cell lung cancer cell line DMS-79 (human SCLC ATCC # CRL-2049).

In other embodiments, V_HAnd/or V_LThe amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence described above. Has and aboveV of the sequence_HAnd V_LV of region height (i.e., 80% or more) homology_HAnd V_LAntibodies to the regions can be obtained as follows: mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of a nucleic acid encoding SEQ ID NO: 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60, and then detecting the retained function of the encoded altered antibody (i.e., the function described in (c) and (d) above) using the functional assay described herein.

As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Considering the number of gaps that need to be introduced for optimal alignment between two sequences and the length of each gap, the percent identity between two sequences is a function of the number of identical sites that are common to the two sequences (i.e.,% homology-the number of identical sites/total number of sites x 100). Sequence comparison and determination of percent identity between two sequences can be accomplished using mathematical algorithms as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithms of e.meyers and w.miller (comput.appl.biosci., 4: 11-17(1988)) which have been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J.mol.biol.48: 444-.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" for public database searches, such as identifying related sequences. This search can be performed using Altschul et al (1990) J.mol.biol.215: XBLAST program version 2.0 from 403-10. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gap alignments for comparison purposes, for example, Altschul et al (1997) Nucleic Acids Res.25 (17): 3389 and 3402. When BLAST and gapped BLAST programs are employed, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (see www.ncbi.nlm.nih.gov.)

Antibodies with conservative modifications

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprises a particular amino acid sequence or conservative modifications thereof based on the preferred antibodies described herein (e.g., 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5), and wherein the antibodies retain the desired functional properties of the anti-fucosyl-GM 1 antibodies of the invention. Accordingly, the present invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising the sequences of CDR1, CDR2 and CDR3 and a light chain variable region comprising the sequences of CDR1, CDR2 and CDR3, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 25. 26, 27, 28, 29 and 30 and conservatively modified amino acid sequences thereof;

(b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 43. 44, 45, 46, 47 and 48 and conservatively modified amino acid sequences thereof; and is

The antibody exhibits one or more of the following properties:

(c) specifically bind to fucosyl-GM 1; and

In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs: 19. 20, 21, 22, 23 and 24 and conservatively modified amino acid sequences thereof; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from SEQ id nos: 37. 38, 39, 40, 41 and 42 and conservatively modified amino acid sequences thereof. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 13. 14, 15, 16, 17 and 18 and conservatively modified amino acid sequences thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 31. 32, 33, 34, 35 and 36 and conservatively modified amino acid sequences thereof.

The term "conservative sequence modification" as used herein refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those that replace an amino acid residue with one having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, one or more amino acid residues within a CDR region of an antibody of the invention can be substituted for other amino acid residues from the same side chain family, and the function retained by the altered antibody (i.e., the function described in (c) and (d) above) can be detected using the functional assays described herein.

Antibodies that bind to the same epitope as the anti-fucosyl-GM 1 antibodies of the invention

In another embodiment, the present invention provides a phase wherein any of the fucosyl-GM 1 monoclonal antibodies of the present invention binds to fucosyl-GM 1An antibody that is homoepitopic (i.e., an antibody that is capable of cross-competing with any monoclonal antibody of the invention for binding to fucosyl-GM 1). In a preferred embodiment, the reference antibody used in the cross-competition study may be monoclonal antibody 5B1 (having V as shown in SEQ ID NOS: 1 and 7, respectively)_HAnd V_LSequence), or monoclonal antibody 5B1a (having the amino acid sequences shown in SEQ id nos: v shown in 2 and 8_HAnd V_LSequence), or monoclonal antibody 7D4 (having the amino acid sequences as set forth in SEQ ID NOs: v shown in FIGS. 3 and 9_HAnd V_LSequence), or monoclonal antibody 7E4 (having the amino acid sequences as set forth in SEQ ID NOs: v shown by 4 and 10_HAnd V_LSequence), or monoclonal antibody 13B8 (having the amino acid sequences as set forth in SEQ ID NOs: v shown in FIGS. 5 and 11_HAnd V_LSequence), or monoclonal antibody 18D5 (having the amino acid sequences as set forth in seq id NOs: v shown in FIGS. 6 and 12_HAnd V_LSequence). These cross-competing antibodies can be identified by their ability to cross-compete with 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5 in a standard fucosyl-GM 1 binding assay. For example, BIAcore analysis, ELISA assays, or flow cytometry analysis can be used to demonstrate cross-competition with the antibodies of the invention. The ability of the test antibody to inhibit binding of, for example, 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5 to fucosyl-GM 1 demonstrates that the test antibody can compete with binding of 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5 to fucosyl-GM 1, and thus bind to the same epitope on fucosyl-GM 1 as 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D 5. In a preferred embodiment, the antibody that binds to the same epitope on fucosyl-GM 1 as 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5 is a human monoclonal antibody. These human monoclonal antibodies can be prepared and isolated as described in the examples.

Engineered antibodies and modified antibodies

The antibodies of the invention further may be utilized with one or more of the V disclosed herein_HAnd/or V_LAntibodies of sequence are prepared as starting materials to construct a modified antibody that may have altered properties compared to the starting antibody. Can be modified by modifying one or both variable regions (i.e., V)_HAnd/or V_L) For example, one or more residues within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, antibodies may be engineered by modifying residues within the constant region, for example to alter the effector function of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with a target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector as follows: the expression vector contains CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al (1998) Nature 332: 323-327; Jones, P. et al (1986) Nature 321: 522-525; Queen, C. et al (1989) Proc. Natl. Acad. Sci. U.S. A.86: 10029-10033; Winter U.S. Pat. No.5,225,539, and Queen et al U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the invention is directed to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, the CDR1, CDR2 and CDR3 sequences respectively comprising an amino acid sequence selected from the group consisting of seq id NOs: 13. 14, 15, 16, 17 and 18, SEQ ID NO: 19. 20, 21, 22, 23 and 24, and SEQ ID NO: 25. 26, 27, 28, 29 and 30, and comprising a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, the CDR1, CDR2 and CDR3 sequences comprising amino acid sequences selected from SEQ ID NOs: 31. 32, 33, 34, 35 and 36, SEQ ID NO: 37. 38, 39, 40, 41 and 42, and SEQ ID NO: 43. 44, 45, 46, 47 and 48. Thus, these antibodies contain the V of monoclonal antibody 5B1, 5B1a, 7D4, 7E4, 13B8 or 18D5_HAnd V_LCDR sequences, but may contain framework sequences different from those of these antibodies.

These framework sequences can be obtained from public DNA databases or published references comprising germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in: "VBase" human germline sequence database (available from the Internet)www.mrc-cpe.cam.ac.uk/vbaseObtained), and Kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, u.s.department of Health and Human Services, NIH publication No. 91-3242; tomlinson, I.M. et al (1992) "the reperture of Human Germine V_HSequences Reveals about Fifty Groups of V_HFragments with Different changeable Loops "J.mol.biol.227: 776-798; and Cox, J.P.L.et al (1994) "A Directory of Human Germ-line V_HSegments variables a StrongBias in the hair Usage "Eur.J. Immunol.24: 827-836; the contents of which are incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7HuMAb mice can be obtained according to the following Genbank accession numbers: 1-69(NG _0010109, NT _024637 and BC070333), 3-33(NG _0010109 and NT _024637), and 3-7(NG _0010109 and NT _ 024637). As another example, the following heavy chain germline sequences found in HCo12HuMAb mice can be obtained according to the Genbank accession numbers as follows: 1-69(NG _0010109, NT _024637 and BC070333), 5-51(NG _0010109 and NT _024637), 4-34(NG _0010109 and NT _024637), 3-30.3 and 3-23(AJ 406678).

Preferred framework sequences for use in antibodies of the invention are similar in structure to the framework sequences used by selected antibodies of the invention, e.g., similar to V used with preferred monoclonal antibodies of the invention_H3-48 framework sequences (SEQ ID NO: 61) and/or V_KThe L15 framework sequence (SEQ ID NO: 62). V_HCDR1, CDR2 and CDR3 sequences and V_KThe CDR1, CDR2 and CDR3 sequences may be grafted onto framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences may be grafted onto framework regions having the same sequence as the germline sequenceTo framework regions containing one or more mutations. For example, it has been found that in some instances, it is advantageous to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Another type of variable region modification is mutation V_HAnd/or V_KAmino acid residues within the CDR1, CDR2, and/or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, effects on antibody binding, or other functional properties of interest, which can be assessed using in vitro or in vivo assays described herein and provided in the examples. Preferably conservative modifications (as described above) are introduced. These mutations may be amino acid substitutions, additions or deletions, but substitutions are preferred. Furthermore, typically no more than 1, 2,3, 4 or 5 residues within a CDR region are altered.

Thus, in another embodiment, the invention provides an isolated anti-fucosyl-GM 1 monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) v_HA CDR1 region comprising an amino acid sequence selected from SEQ ID NOs: 13. 14, 15, 16, 17 and 18, or a sequence identical to SEQ ID NO: 13. 14, 15, 16, 17 and 18 with 1, 2,3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence; (b) v_HA CDR2 region comprising an amino acid sequence selected from SEQ ID NOs: 19. 20, 21, 22, 23 and 24, or an amino acid sequence substantially identical to SEQ ID NO: 19. 20, 21, 22, 23 and 24 have an amino acid sequence having 1, 2,3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence; (c) v_HA CDR3 region comprising an amino acid sequence selected from SEQ ID NOs: 25. 26, 27, 28, 29 and 30, or a sequence identical to SEQ ID NO: 25. 26, 27, 28, 29 and 30 with 1, 2,3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence; (d) v_KA CDR1 region comprising an amino acid sequence selected from SEQ ID NOs: 31. 32, 33, 34, 35 and 36, or an amino acid sequence substantially identical to SEQ ID NO: 31. 32, 33, 34, 35 and 36 have 1, 2,3, 4 or 5 amino acid substitutions, deletions compared toAmino acid sequences that are missing or added; (e) v_KA CDR2 region comprising an amino acid sequence selected from SEQ ID NOs: 37. 38, 39, 40, 41 and 42, or an amino acid sequence substantially identical to SEQ ID NO: 37. 38, 39, 40, 41 and 42 have an amino acid sequence having 1, 2,3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence; and (f) V_KA CDR3 region comprising an amino acid sequence selected from SEQ ID NOs: 43. 44, 45, 46, 47 and 48, or an amino acid sequence substantially identical to SEQ ID NO: 43. 44, 45, 46, 47 and 48 have amino acid sequences with 1, 2,3, 4 or 5 amino acid substitutions, deletions or additions compared to the corresponding amino acid sequence.

Engineered antibodies of the invention include, for example, V against antibodies for improved antibody properties_HAnd/or V_KAntibodies in which the framework residues are modified. Such framework modifications are typically made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues to the corresponding germline sequence. More particularly, an antibody undergoing somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. These residues can be identified by comparing the antibody framework sequences to the germline sequence from which the antibody was derived.

For example, for 7E4, V_HAmino acid residue #11 (within FR 1) of (1) is serine, and in the corresponding V_H3-48 the residue in the germline sequence is leucine. To restore the framework sequences to their germline configuration, the somatic mutations can be "back-mutated" to germline sequences (e.g., V for 7E 4) by, for example, site-directed mutagenesis or PCR-mediated mutagenesis_HResidue #11 of FR1 may be "back-mutated" from serine to leucine).

Another example is for 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5, V_HAmino acid residue #16 (within FR 1) of (1) is glutamic acid, and in the corresponding V_HThe residue in the 3-48 germline sequences is glycine. To restore the framework sequence to its germline configuration, for example, V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 can be substituted_HResidue #16 of (a) is "back-mutated" from glutamic acid to glycine. Such "back-mutated" antibodies are also encompassed by the present invention.

Another example is for 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5, V_HIs valine at amino acid residue #23 (within FR 1) of (A), and is in the corresponding V_H3-48 the residue in the germline sequence is alanine. To restore the framework sequence to its germline configuration, for example, V of 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 can be substituted_HResidue #23 of (a) is "back-mutated" from valine to alanine. Such "back-mutated" antibodies are also encompassed by the present invention.

Another example is, for 7D4, V_HIs valine at amino acid residue #24 (within FR 1) of (A), and is in the corresponding V_H3-48 the residue in the germline sequence is alanine. To restore the framework sequence to its germline configuration, for example, V of 7D4 can be_HResidue #24 of (a) is "back-mutated" from valine to alanine. Such "back-mutated" antibodies are also encompassed by the present invention.

Another example is, for 13B8, V_HAmino acid residue #29 (within FR 1) of (I) is leucine, and in the corresponding V_H3-48 the residue in the germline sequence is phenylalanine. To restore the framework sequence to its germline configuration, V of 13B8, for example, can be substituted_HResidue #29 of (a) was "back-mutated" from leucine to phenylalanine. Such "back-mutated" antibodies are also encompassed by the present invention.

Another example is, for 7D4, 13B8 and 18D5, V_HIs isoleucine at amino acid residue #48 (within FR 2) of (4), and is in the corresponding V_HThe residue in the 3-48 germline sequence is valine. To restore the framework sequence to its germline configuration, for example, V's of 7D4, 13B8, and 18D5 can be combined_HResidue #48 (residue #13 within FR 2) was "back-mutated" from isoleucine to valine. Such "back-mutated" antibodies are also encompassed by the present invention. Another example is, for 7D4 and 18D5, V_HIs serine at amino acid residue #84 (within FR 3) of (4), and is in the corresponding V_H3-48 the residue in the germline sequence is asparagine. To restore the framework sequence to its germline configuration, for example,v of 7D4 and 18D5 can be adjusted_HResidue #84 (residue #18 within FR 3) was "back-mutated" from serine to asparagine. Such "back-mutated" antibodies are also encompassed by the present invention.

Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also called "deimmunization" and is described in detail in U.S. patent publication No. 20030153043 to Carr et al.

In addition to, or as an alternative to, modifications made within the framework or CDR regions, the antibodies of the invention may also be engineered to include modifications within the Fc region, typically in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention may also be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), or modified to alter glycosylation, to alter one or more functional properties of the antibody. These embodiments are described in detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This process is described in detail in U.S. Pat. No.5,677,425 to Bodmer et al. The number of cysteines in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody binds less well to staphylococcal protein a (SpA) than to SpA as compared to the native Fc-hinge domain. This method is described in detail in U.S. Pat. No.6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various methods may be used. For example, as described in U.S. Pat. No.6,277,375 to Ward, one or more of the following mutations may be introduced: T252L, T254S, T256F. Alternatively, to increase biological half-life, the antibody may be altered in the CH1 or CL region to contain salvage receptor binding epitopes from both loops of the CH2 domain of the IgG Fc region, as described in U.S. Pat. Nos.5,869,046 and 6,121,022 to Presta et al.

In other embodiments, the Fc region is altered by substituting at least one amino acid residue for a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322 may be substituted with a different amino acid residue such that the affinity of the antibody for the effector ligand is altered, but the antigen binding ability of the parent antibody is retained. The effector ligand whose affinity is altered may be, for example, an Fc receptor or the C1 component of complement. This method is described in more detail in U.S. Pat. Nos.5,624,821 and 5,648,260 to Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be substituted with a different amino acid residue such that C1q binding of the antibody is altered and/or Complement Dependent Cytotoxicity (CDC) is reduced or eliminated. This method is described in more detail in U.S. Pat. No.6,194,551 to Idusogene et al.

In another embodiment, one or more of the amino acid residues in amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to fix complement. The method is further described in PCT publication WO94/29351 by Bodmer et al.

In another embodiment, to increase the ability of an antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of an antibody for fey receptors, the Fc region is modified by modifying one or more amino acids at the following sites: 238. 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This method is further described in PCT publication WO00/42072 to Presta. Furthermore, the binding sites on human IgG1 for Fc γ RI, Fc γ RII, Fc γ RIII and FcRn have been mapped and variants with improved binding have been described (see, Shiels, R.L. et al (2001) J.biol.chem.276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to Fc γ RIII. In addition, the following combination mutants showed improved binding to Fc γ RIII: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In another embodiment, the glycosylation of the antibody is altered. For example, an antibody can be made that is aglycosylated (i.e., the antibody lacks glycosylation). For example, glycosylation can be altered in order to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation may increase the affinity of the antibody for the antigen. Such methods are described in more detail in U.S. Pat. Nos.5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered glycosylation patterns can be made, such as low fucosylated antibodies with reduced numbers of fucosyl residues, or antibodies with equally divided GlcNac structures increased. This altered glycosylation pattern has been shown to improve the ADCC ability of the antibody. Such carbohydrate modification can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art as being capable of use as host cells in which to expressFor example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8(α (1, 6) fucosyltransferase gene), thus antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their carbohydrates.Ms 704, Ms705, and Ms709FUT8^-/-Cell lines are generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two alternative vectors (see Yamane et al, U.S. patent application No.20040110704 and Yamane-Ohnuki et al (2004) Biotechnol Bioeng 87: 614-22). Another example is that EP1,176,195 by Hanai et al describes cell lines with a functionally disrupted FUT8 gene, the FUT8 gene encodes a fucosyltransferase, and the antibody expressed in such cell lines is low fucosylated by reducing or eliminating the enzyme associated with the 1,6 bond of the FUT α. Hanai et al also describes cell lines with low or no fucokinase activity for addition to N-acetylglucosamine binding to the Fc region of the antibody, e.g. PCT 03/035835 by rat myeloma lines 2/0(ATCC 1662) Presta myeloma lines, e.g. PCT 03/035835 by the engineering of fucosyltransferase lines with increased fucosyltransferase expression in cells (e.g. the cell lines with the fucose-glycosyltransferase binding to the Fc region of the antibody, e.g. the cell lines with reduced fucosyltransferase expression of the enzyme by linking the fucose-glycosyltransferase (see PCT-17. A-Na-NO: CHO, CHO-17, U.g. the engineered glycoprotein expressed by the cell lines of the antibody, I-Na-acetyl transferase, U.g. the antibody, U.7, No. the antibody, No.5, No. NO.7, No. 15, No.7, No. 15, No.7, No. 4. A-9) modified by the antibody, No. 7.

Another modification to the antibodies described herein that the invention relates to is PEGylation. For example, to increase the biological (e.g., serum) half-life of an antibody, the antibody can be pegylated. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed by acylation or alkylation with a reactive PEG molecule (or similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any PEG form used to derivatize other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods of PEGylating proteins are well known in the art and may be used with the antibodies of the invention. See, for example, EP 0154316 to Nishimura et al and EP 0401384 to Ishikawa et al.

Antibody engineering method

As described above, V having the disclosure herein can be utilized_HAnd V_Kanti-fucosyl-GM 1 antibodies of sequence by modification of V_HAnd/or V_KThe sequence or constant region linked thereto, giving rise to novel anti-fucosyl-GM 1 antibodies. Thus, in another aspect of the invention, the structural features of an anti-fucosyl-GM 1 antibody of the invention (e.g., 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5) are exploited to generate a structurally related anti-fucosyl-GM 1 antibody that retains at least one functional property of the antibody of the invention, such as binding to fucosyl-GM 1. As described above, for example, one or more CDR regions of 5B1, 5B1a, 7D4, 7E4, 13B8, or 18D5 or mutations thereof may be recombined in combination with known framework regions and/or other CDRs to produce additional recombinantly engineered anti-fucosyl-GM 1 antibodies of the invention. Other modification types include those described in the section above. Starting materials for the engineering process are one or more of V provided herein_HAnd/or V_KA sequence, or one or more CDR regions thereof. In order to produce an engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) a polypeptide having one or more of the V provided herein_HAnd/or V_KAn antibody having the sequence, or one or more CDR regions thereof. But rather in the sequenceThe information contained serves as starting material, generating a "second generation" sequence derived from the original sequence, which is then prepared and expressed as a protein.

Thus, in another embodiment, the invention provides a method of making an anti-fucosyl-GM 1 antibody, comprising:

(a) providing: (i) a heavy chain variable region antibody sequence comprising a sequence selected from SEQ ID NOs: 13. 14, 15, 16, 17 and 18, a CDR1 sequence selected from SEQ ID NOs: 19. 20, 21, 22, 23 and 24, and/or a CDR2 sequence selected from SEQ ID NOs: 25. 26, 27, 28, 29 and 30; and/or (ii) a light chain variable region antibody sequence comprising a sequence selected from SEQ ID NOs: 31. 32, 33, 34, 35 and 36, a CDR1 sequence selected from SEQ ID NOs: 37. 38, 39, 40, 41 and 42, and/or a CDR2 sequence selected from SEQ ID NOs: 43. CDR3 sequences of 44, 45, 46, 47 and 48;

(b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence, thereby producing at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

The altered antibody sequences can be prepared and expressed using standard molecular biology techniques.

Preferably, the antibody encoded by the altered antibody sequence retains one, some or all of the functional properties of the anti-fucosyl-GM 1 antibody described herein, including but not limited to:

(a) the antibody is expressed as 1 × 10^-7K of M or less_DBinding to fucosyl-GM 1;

The functional properties of the altered antibody can be assessed using standard assays (e.g., flow cytometry, binding assays) used in the art and/or described herein (as described in the examples).

In certain embodiments of the methods of engineering antibodies of the invention, mutations may be introduced randomly or selectively along all or part of the anti-fucosyl-GM 1 antibody coding sequence, and the resulting modified anti-fucosyl-GM 1 antibodies may be screened for binding activity and/or other functional properties as described herein. Methods of mutagenesis have been described in the art. For example, PCT publication WO02/092780 to Short describes methods for generating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. PCT publication WO03/074679 by Lazar et al also describes methods for optimizing the physiochemical properties of antibodies using computational screening methods.

Nucleic acid molecules encoding the antibodies of the invention

Another aspect of the invention relates to a nucleic acid molecule encoding an antibody of the invention. The nucleic acid may be present in whole cells, cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when separated and purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See, F.Ausubel et al ed. (1987) Current Protocols in Molecular Biology, Greene publishing and Wiley Interscience, New York. The nucleic acids of the invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cdnas encoding the light and heavy chains of antibodies prepared by the hybridomas can be obtained using standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the libraries.

Preferred nucleic acid molecules of the invention are V encoding the 5B1, 5B1a, 7D4, 7E4, 13B8 or 3C4 monoclonal antibodies_HAnd V_LA nucleic acid molecule of sequence. V encoding 5B1, 5B1a, 7D4, 7E4, 13B8 and 3C4_HThe DNA sequences of the sequences are shown in SEQ ID NO: 49. 50, 51, 52, 53 and 54. V encoding 5B1, 5B1a, 7D4, 7E4, 13B8 and 3C4_LThe DNA sequences of the sequences are set forth in SEQ ID NOs: 55. 56, 57, 58, 59 and 60.

Once the code V is obtained_HAnd V_LSegmented DNA fragments, which can be further manipulated by standard recombinant DNA techniques, such as conversion of the variable region gene to a full-length antibody chain gene, Fab fragment gene or scFv gene. In these operations, V will be encoded_LOr V_HIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that two DNA segments are linked together such that the amino acid sequences encoded by the two DNA segments remain in reading frame.

By encoding V_HIs operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3) and isolated DNA encoding V_HDNA of the region is converted to the full-length heavy chain gene. The sequence of the Human heavy chain constant region gene is well known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of immunology interest, fifth edition, U.S. department of Health and Human Services, NIH publication No. 91-3242), and DNA fragments including these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, V is encoded_HMay be operably linked to another DNA molecule encoding only the constant region of heavy chain CH 1.

By encoding V_LIs operably linked to another DNA molecule encoding a light chain constant region CLThe separate codes V can be combined_LThe DNA of the region is converted to the full-length light chain gene (as well as the Fab light chain gene). The sequence of the Human light chain constant region gene is well known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of immunologic Interest, fifth edition, U.S. department of Health and Human Services, NIH publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is most preferably a kappa constant region.

To generate the scFv gene, V will be encoded_HAnd V_LWith a DNA fragment encoding a flexible linker, e.g.an amino acid sequence (Gly)₄-Ser)₃Is operably linked such that V_HAnd V_LThe sequence may be expressed as a continuous single-chain protein, V_LAnd V_HThe regions are linked by this flexible linker (see, e.g., Bird et al (1988) Science 242: 423-.

Generation of monoclonal antibodies of the invention

The monoclonal antibodies (mabs) of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody methods, e.g., Kohler and Milstein (1975) Nature 256: 495 by standard somatic hybridization techniques. Although somatic cell hybridization techniques are preferred, in principle, other techniques for preparing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murine system. The generation of hybridomas with mice is a well established procedure. Immunization procedures and techniques for isolating immunized splenocytes for fusion are well known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also well known.

The chimeric or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to generate chimeric antibodies, murine variable regions can be joined to human constant regions using methods well known in the art (see, e.g., U.S. Pat. No.4,816,567 to Cabilly et al). To generate humanized antibodies, murine CDR regions can be inserted into human frameworks using methods well known in the art (see, e.g., U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

In a preferred embodiment, the antibodies of the invention are human monoclonal antibodies. Such anti-fucosyl-GM 1 human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying part of the human immune system rather than the mouse system. These transgenic and transchromosomal mice are included herein referred to as HuMab mice and KM mice, respectively^TMAnd is herein generally referred to as a "human Ig mouse".

HuMab mice(Metarex, Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, and a targeted mutation that inactivates the endogenous mu and kappa chain loci (see, e.g., Lonberg et al (1994) Nature368 (6474): 856-. Thus, the mouse shows reduced mouse IgM or kappa expression and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation, thereby producing a high affinity human IgG kappa monoclonal antibody (Lonberg, N. et al (1994), supra; reviewed in Lonberg, N. (1994) handbook Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern.Rev.Immunol.Vol.13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y.Acad.Sciad.764: 536-. The preparation and use of HuMab mice, as well as the genomic modifications carried by the mice, are described in detail in the following literature: taylor, L. et al (1992) Nucleic Acids Research 20: 6287-6295; chen, J, et al(1993) International Immunology 5: 647-656; tuaillon et al (1993) Proc. Natl. Acad. Sci USA 90: 3720-3724; choi et al (1993) Nature Genetics 4: 117-; chen, J. et al (1993) EMBO J.12: 821-830; tuaillon et al (1994) J.Immunol.152: 2912-2920; taylor, L, et al (1994) International Immunology 6: 579-; and fisherworld, d. et al (1996) nature biotechnology 14: 845-851, the contents of which are incorporated herein by reference in their entirety. See, further, U.S. Pat. Nos.5,545,806 to Lonberg and Kay; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,789,650, respectively; 5,877,397, respectively; 5,661,016, respectively; 5,814, 318; 5,874,299, respectively; and 5,770,429; and U.S. patent No.5,545,807 to Surani et al; PCT publication Nos. WO92/03918, WO93/12227, WO94/25585, WO97/13852, WO98/24884 and WO99/45962 to Lonberg and Kay; and PCT publication No. WO01/14424 by Korman et al.

In another embodiment, the human antibodies of the invention can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes. Such mice are referred to herein as "KM mice^TM", are described in detail in PCT publication WO02/43478 to Ishida et al.

Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-fucosyl-GM 1 antibodies of the invention. For example, an alternative transgene system known as xenomoue (Abgenix, Inc.) may be used, such mice being described in, for example, U.S. patent nos.5,939,598 to Kucherlapati et al; 6,075,181; 6,114,598, respectively; 6,150,584 and 6,162,963.

Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-fucosyl-GM 1 antibodies of the invention. For example, mice carrying human heavy chain transchromosomes and human light chain transchromosomes, referred to as "TC mice" may be used; this mouse is described in Tomizuka et al (2000) proc.natl.acad.sci.usa 97: 722-727. Furthermore, bovine carrying human heavy and light chain transchromosomes (Kuroiwa et al (2002) Nature Biotechnology 20: 889-894) have been described in the art, which can be used to produce the anti-fucosyl-GM 1 antibodies of the invention.

The human monoclonal antibodies of the invention can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See, for example, U.S. Pat. Nos.5,223,409 to Ladner et al; 5,403,484; and 5,571,698; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al; U.S. patent nos.5,969,108 and 6,172,197 to McCafferty et al; and U.S. Pat. No.5,885,793 to Griffiths et al; 6,521,404; 6,544,731, respectively; 6,555,313, respectively; 6,582,915, and 6,593,081.

The human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells are reconstituted and thus capable of producing human antibody responses upon immunization. Such mice are described, for example, in U.S. patent nos.5,476,996 and 5,698,767 to Wilson et al.

Immunization of human Ig mice

When human antibodies of the invention are generated using human Ig mice, they can be generated according to Lonberg, N.et al (1994) Nature368 (6474): 856-859; fishwild, D. et al (1996) Nature Biotechnology 14: 845-; and PCT publications WO98/24884 and WO01/14424, the mice were immunized with purified or enriched preparations of fucosyl-GM 1 antigen and/or recombinant fucosyl-GM 1 or fucosyl-GM 1 fusion proteins. Preferably, the mice are 6-16 weeks old at the time of the first infusion. For example, human Ig mice can be immunized intraperitoneally with a preparation (5-50 μ g) of purified or recombinant fucosyl-GM 1 antigen.

The detailed procedure for the production of fully human monoclonal antibodies to fucosyl-GM 1 is described in example 1 below. Experience with various antigen accumulations demonstrated that transgenic mice respond when initially immunized Intraperitoneally (IP) with antigen in freund's complete adjuvant, followed by intraperitoneal immunization with antigen in freund's incomplete adjuvant every other week (up to six times total). However, adjuvants other than Freund's adjuvant have been found to be effective. In addition, the hairWhole cells are highly immunogenic in the absence of adjuvant. The immune response was monitored during the course of the immunization protocol using plasma samples obtained from retroorbital bleeds. Plasma can be screened by ELISA (as described below), using mice with sufficient titers of anti-fucosyl-GM 1 human immunoglobulin for fusion. Mice were boosted intravenously with antigen, sacrificed 3 days later and spleens removed. It is expected that 2-3 fusions may be required for each immunization. Each antigen was used to immunize 6-24 mice. In general, strains HCo7 and HCo12 are used. In addition, the HCo7 and HCo12 transgenes can be crossed to produce a mouse with two different human heavy chain transgenes (HCo7/HCo 12). Alternatively or additionally, as described in example 1, KM mice may be used^TMAnd (4) strain.

Preparation of hybridomas producing human monoclonal antibodies of the present invention

For the preparation of hybridomas producing human monoclonal antibodies of the present invention, spleen cells and/or lymph node cells are isolated from immunized mice and fused with a suitable immortalized cell line (e.g., a mouse myeloma cell line). hybridomas selected based on the production of antigen-specific antibodies, for example, a single cell suspension of splenic lymphocytes from immunized mice can be fused with one-sixth number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL1580) using 50% PEG, or alternatively, a single cell suspension of splenic lymphocytes from immunized mice can be fused using an electric field-based electrofusion method using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, Md.) cells are fused at about 2 × 10⁵Was inoculated in flat-bottomed microtiter plates followed by one week incubation in DMEM high concentration glucose medium containing L-glutamine and sodium pyruvate (Mediatech, Inc., Herndon, Va.), and also containing 20% fetal bovine serum (Hyclone, Logan, UT), 18% P388DI conditioned medium, 5% Origen hybridoma cloning factor (BioVeris, Gaithersburg, Va), 4mM L-glutamine, 5mM HEPES, 0.055mM β -mercaptoethanol, 50 units/ml penicillin, 50mg/ml streptomycin, and 1 × hypoxanthine-aminopterin-thymidine (M.) (Va.) (A)HAT) medium (Sigma; HAT was added 24 hours after fusion). One week later, cells were cultured in medium in which HAT was replaced with HT. Each well was then screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth has occurred, the medium can be observed, usually after 10-14 days. Antibody secreting hybridomas were replated and screened again, and if still positive for human IgG, monoclonal antibodies could be subcloned at least twice by limiting dilution. The stable subclones were then cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas were grown in two liter rotary shake flasks for monoclonal antibody purification. The supernatant was filtered, concentrated, and then subjected to affinity chromatography using protein a-sepharose (Pharmacia, Piscataway, n.j.). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution was changed to PBS and the concentration was determined from OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and stored at-80 ℃.

Preparation of transfectomas producing the monoclonal antibodies of the invention

Antibodies of the invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) science 229: 1202).

For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the antibody of interest) and inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody genes are linked into a vector such that transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody genes. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., complementary restriction sites on the antibody gene fragment are ligated to the vector, or blunt-ended if no restriction sites are present). By insertion into an expression vector which already encodes the heavy and light chain constant regions of the desired isotype, V is made_HC in segments and vectors_HThe segments are operatively connected, and V_KC in segments and vectors_LThe segments are operably linked and the light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame with the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than an immunoglobulin).

In addition to the antibody chain gene, the recombinant expression vector of the present invention also carries regulatory sequences that control the expression of the antibody chain gene in a host cell. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene expression technology. methods in Enzymology185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the choice of regulatory sequences, will depend on factors such as the choice of host cell to be transformed, the level of protein expression desired, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and polyoma viruses. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the beta-globin promoter, can be used. In addition, the regulatory elements may also consist of sequences from different sources, such as the SR α promoter system, which contains the sequence from the SV40 early promoter and the long terminal repeat of the human T cell leukemia virus type 1 (Takebe, Y., et al (1988) mol.cell.biol.8: 466-472).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may also carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 to Axel et al). For example, selectable marker genes typically confer drug resistance, e.g., G418, hygromycin or methotrexate resistance, to a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells for methotrexate selection/amplification) and the neo gene (for G418 selection).

To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" of various forms including commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in various techniques, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection. While it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, most preferably mammalian host cells, because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to fail to produce high yields of active antibodies (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expression of recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (including Urlaub and Chasin, (1980) DHFR-CHO cells as described in Proc. Natl. Acad. Sci. USA77: 4216-. In particular, another preferred expression system for NSO myeloma cells is the GS gene expression system disclosed in WO87/04462, WO89/01036 and EP338,841. When a recombinant expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed in the host cell, or more preferably, for a period of time sufficient for the antibody to be secreted into the medium in which the host cell is grown. Standard protein purification methods can be used to recover the antibody from the culture medium.

Characterization of antibody binding to antigen

Binding of the antibodies of the invention to fucosyl-GM 1 is detected, for example, by standard ELISA. Briefly, microtiter plates were coated with 0.25. mu.g/ml of purified fucosyl-GM 1 in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of the antibody (e.g., dilutions of plasma from fucosyl-GM 1 immunized mice) were added to each well and incubated for 1-2 hours at 37 ℃. The plates were washed with PBS/Tween and then incubated with a second reagent conjugated to alkaline phosphatase (e.g., goat anti-human IgG Fc specific polyclonal reagent for human antibodies) at 37 ℃ for 1 hour. After washing, the plates were developed with pNPP substrate (1mg/ml) and analyzed at OD 405-650. Preferably, the fusion is performed with the mouse showing the highest titer.

The ELISA assay described above can also be used to screen for hybridomas that exhibit positive reactivity with fucosyl-GM 1 immunogen. Hybridomas that bound fucosyl-GM 1 with high affinity were subcloned and further characterized. One clone that retained the reactivity of the mother cells was selected from each hybridoma (by ELISA), and 5-10 vial cell banks were prepared and stored at-140 ℃ for antibody purification.

Selected hybridizations for purification of anti-fucosyl-GM 1 antibodiesTumors were grown in two liter rotary shake flasks for monoclonal antibody purification. The supernatant was filtered and concentrated, followed by affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). The eluted IgG was checked by gel electrophoresis and high performance liquid chromatography to ensure purity. Buffer solution was changed to PBS and extinction coefficient according to OD using 1.43₂₈₀The concentration is determined. Monoclonal antibodies were split into aliquots and stored at-80 ℃.

To determine whether the selected anti-fucosyl-GM 1 monoclonal antibodies bound to a unique epitope, each antibody was biotinylated using a commercially available reagent (Pierce, Rockford, IL). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using fucosyl-GM 1 coated ELISA plates as described above. Binding of biotinylated mabs can be detected using a streptavidin-alkaline phosphatase probe.

To determine the isotype of the purified antibody, an isotype ELISA may be performed with reagents specific for the particular isotype of antibody. For example, to determine the isotype of human monoclonal antibodies, wells of microtiter plates were coated with 1. mu.g/ml of anti-human immunoglobulin overnight at 4 ℃. After blocking with 1% BSA, the plates were reacted with 1. mu.g/ml or less of the monoclonal antibody to be tested or purified isotype control for 1-2 hours at ambient temperature. These wells were then reacted with human IgG1 or human IgM specific alkaline phosphatase conjugated probes. The slab is colored and dispersed as described above.

The reactivity of anti-fucosyl-GM 1 human IgG to the fucosyl-GM 1 antigen can be further examined by Western blotting. Briefly, fucosyl-GM 1 was prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens were transferred to nitrocellulose membranes, blocked with 10% fetal bovine serum, and probed with the monoclonal antibodies to be tested. Binding of human IgG can be detected with anti-human IgG alkaline phosphatase and visualized with BCIP/NBT substrate sheets (Sigma chem.co., st.louis, Mo.).

Immunoconjugates

In another aspect, the invention relates to an anti-fucosyl-GM 1 antibody or fragment thereof conjugated to a therapeutic moiety such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. These conjugates are referred to herein as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is detrimental to a cell (e.g., kills a cell). Examples include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thiophenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine (decarbazine)), alkylating agents (e.g., mechlorethamine, chlorambucil (thioephalorambucil), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthranilones (e.g., daunorubicin (previously known as daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D (previously known as actinomycin), bleomycin, mithramycin and Atramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the invention include duocarmycin, calicheamicin, maytansine, auristatin, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg)^TM(ii) a Wyeth-Ayerst). Examples of therapeutic cytotoxins can be found, for example, in U.S. patents 6548530 and 6281354 and U.S. patent applications US2003/0064984, US2003/0073852 and US 2003/0050331.

Cytotoxins can be conjugated to antibodies of the invention using linker technology used in the art. Examples of types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Alternatively, for example, a linker that is susceptible to cleavage by low pH or by a protease, such as a protease preferentially expressed in tumor tissue, such as cathepsin (e.g., cathepsin B, C, D), within the lysosomal compartment can be selected.

For further discussion of the type of cytotoxin, linkers and methods used to couple therapeutic agents to antibodies, see Saito, g, et al (2003) adv. drug delivery div.rev.55: 199-; trail, p.a. et al (2003) cancer.immunol.immunoher.52: 328-337; payne, G. (2003) Cancer Cell 3: 207-212; allen, t.m. (2002) nat. rev. cancer 2: 750- > 763; patan, i, and Kreitman, R.J, (2002) curr. opin. investig. drugs 3: 1089-; senter, P.D. and Springer, C.J. (2001) adv.drug Deliv.Rev.53: 247-264.

The antibodies of the invention may also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine¹³¹Indium, indium¹¹¹Yttrium, yttrium⁹⁰And lutetium¹⁷⁷. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are available as commercial products, including Zevalin^TM(IDEC Pharmaceuticals) and Bexxar^TM(Corixa Pharmaceuticals) and can be prepared using the antibodies of the invention using similar methods.

The antibody conjugates of the invention may be used to alter a particular biological response, and the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins include, for example: toxins or active fragments thereof having enzymatic activity, such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; proteins, such as tumor necrosis factor or interferon-gamma; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques For coupling such therapeutic moieties to Antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", inner Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (ed.), pp.243-56(AlanR.Liss, Inc.1985); hellstrom et al, "Antibodies For Drug Delivery", in ControlledDrug Delivery (2nd Ed.), Robinson et al (Ed.), pp.623-53(Marcel Dekker, Inc.1987); thorpe, "Antibody Carriers Of Cytotoxin Agents In Cancer Therapy: a Review ", Inmonoclonal Antibodies' 84: biological And Clinical Applications, Pinchera et al (ed.), pp.475-506 (1985); "Analysis, Results, And" Future therapeutic Of therapeutic Use Of radioactive Antibody In Cancer Therapy ", In monoclonal antibodies For Cancer Detection And Therapy, Baldwin et al (ed.), pp.303-16(academic Press1985), And Thorpe et al," The Preparation Of Antibody proteins Of Antibody-Toxin Conjugates, "Immunol.Rev., 62: 119-58(1982).

Bispecific molecules

In another aspect, the invention relates to a bispecific molecule comprising an anti-fucosyl-GM 1 antibody or a fragment thereof according to the invention. The antibodies of the invention or antigen-binding portions thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), thereby generating a bispecific molecule that can bind to at least two different binding sites or target molecules. The antibodies of the invention may in fact be derivatized or linked to more than one other functional molecule, thereby generating multispecific molecules that can bind to more than two different binding sites and/or target molecules; such multispecific molecules are also included within the term "bispecific molecules" as used herein. To produce a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, etc.) to one or more other binding molecules, such as other antibodies, antibody fragments, peptides, or binding mimetics, to provide a bispecific molecule.

Accordingly, the present invention includes bispecific molecules having at least a first binding specificity for fucosyl-GM 1 and a second binding specificity for a second epitope of interest. In a particular embodiment of the invention, the second epitope of interest is an Fc receptor, such as the human fcyri (CD64) or the human fcalpha receptor (CD 89). Thus, the invention includes bispecific molecules that bind both to effector cells expressing Fc γ R or Fc α R, such as monocytes, macrophages or polymorphonuclear cells (PMNs), and to target cells expressing fucosyl-GM 1. These bispecific molecules direct fucosyl-GM 1-expressing cells to effector cells and trigger Fc receptor-mediated effector cell activities such as phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion production by fucosyl-GM 1-expressing cells.

In one embodiment of the invention wherein the bispecific molecule is a multispecific molecule, the molecule may comprise a third binding specificity in addition to the anti-Fc binding specificity and the anti-fucosyl-GM 1 binding specificity. In one embodiment, the third binding specificity is an anti-Enhancement Factor (EF) moiety, e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby enhancing an immune response against a target cell. An "anti-enhancer moiety" can be an antibody, functional antibody fragment, or ligand that binds to a particular molecule, such as an antigen or receptor, resulting in an enhanced effect of the binding determinant on the Fc receptor or target cell antigen. The "anti-enhancer moiety" can bind to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancer moiety may be bound to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancer element moiety can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an enhanced immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise at least one antibody or antibody fragment thereof as binding specificity, including, for example, Fab ', F (ab')₂Fv, or single-chain Fv. The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or a single chain construct, as described in U.S. Pat. No.4,946,778 to Ladner et al, the contents of which are incorporated herein by reference.

In one embodiment, the binding specificity for the Fc γ receptor is provided by a monoclonal antibody whose binding is not blocked by human immunoglobulin g (igg). The term "IgG receptor" as used herein refers to any of the 8 γ -chain genes located on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms, which are divided into 3 Fc γ receptor classes: fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD 16). In a preferred embodiment, the Fc γ receptor is a human high affinity Fc γ RI. Human Fc γ RI is a 72kDa molecule that exhibits high affinity for monomeric IgG (10)⁸-10⁹M^-1)。

The preparation and characterization of certain preferred anti-Fc γ monoclonal antibodies is described by Fanger et al in PCT application WO88/00052 and U.S. Pat. No.4,954,617, the contents of which are incorporated herein by reference in their entirety. These antibodies bind to an epitope of Fc γ RI, Fc γ RII or Fc γ RIII at a site different from the Fc γ binding site of the receptor, and thus, their binding is not substantially blocked by physiological levels of IgG. Specific anti-Fc γ RI antibodies useful in the present invention are mAb22, mAb32, mAb44, mAb62, and mAb 197. The hybridoma producing mAb32 is available from the american type culture collection with ATCC accession number HB 9469. In other embodiments, the anti-Fc γ receptor antibody is a humanized form of monoclonal antibody 22 (H22). Production and characterization of H22 antibodies was described in Graziano, r.f. et al (1995) j.immunol155 (10): 4996 and in PCT publication WO 94/10332. The cell line producing the H22 antibody was deposited with the American type culture Collection under the name HA022CL1 and deposited under accession number CRL 11177.

In other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, such as the Fc- α receptor (Fc α RI (CD89)), which binding is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" includes the gene product of a α -gene located on chromosome 19 (Fc α RI). this gene is known to encode several alternative splice transmembrane isoforms of 55-110 kDa. Fc α RI (CD89) is constitutively expressed on monocytes/macrophages, eosinophils, and neutrophils, but not on non-effector cell populations.Fc α RI has intermediate affinity for both IgA1 and IgA2 (about 5 × 10 IgA)⁷M^-1) This affinity increases after exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al (1996) Critical Reviews in Immunology 16: 423-440.) 4 Fc α RI-specific monoclonal antibodies have been described which are identified as A3, A59, A62 and A77, which bind to Fc α RI outside the IgA ligand binding domain (Monteiro, R.C. et al (1992) J.Immunol.148: 1764).

Fc α RI and Fc γ RI are preferred trigger receptors for bispecific molecules of the invention because they (1) are predominantly expressed on immune effector cells such as monocytes, PMNs, macrophages and dendritic cells; (2) high level expression (e.g., 100,000 cells per cell); (3) are mediators of cytotoxicity (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens directed to them, including autoantigens.

Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules of the invention can be prepared by coupling component binding specificities, such as anti-FcR and anti-fucosyl-GM 1 binding specificities, using methods well known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then coupled to each other. When the binding specificity is a protein or peptide, covalent coupling can be performed using a variety of coupling or crosslinking agents. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenebismaleimide (oPDM), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexyl-1-carboxylate (sulfoo-SMCC) (see, e.g., Karpovsky et al (1984) J.Exp.Med.160: 1686; Liu, MA et al (1985) Proc.Natl.Acad.Sci.USA 82: 8648). Other methods include Paulus (1985) Behring ins. Mitt. No.78, 118-: 81-83 and Glennie et al (1987) J.Immunol.139: 2367 and 2375. Preferred coupling agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co.

When the binding specificities are antibodies, they may be coupled by sulfhydryl bonding of the C-terminal hinge region of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified so that it contains an odd number, preferably 1, of thiol residues prior to coupling.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell when the bispecific molecule is mAb × mAb, mAb × Fab, Fab × F (ab')₂Or ligand × Fab fusion protein, the methods are particularly useful.the bispecific molecules of the invention can be single chain molecules comprising a single chain antibody and one binding determinant, or single chain bispecific molecules comprising two binding determinants the bispecific molecules can comprise at least two single chain molecules methods of making bispecific molecules are described, for example, in U.S. patent 5,260,203, U.S. patent 5,455,030, U.S. patent 4,881,175, U.S. patent 5,132,405, U.S. patent 5,091,513, U.S. patent 5,476,786, U.S. patent 5,013,653, U.S. patent 5,258,498, and U.S. patent 5,482,858.

Binding of a bispecific molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot analysis. Each of these assays typically detects the presence of a particular target protein-antibody complex by applying a labeled reagent (e.g., an antibody) specific for the target complex. For example, FcR-antibody complexes can be detected using enzyme-linked antibodies or antibody fragments that recognize and specifically bind to the antibody-FcR complex. Alternatively, these complexes can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in Radioimmunoassays (RIA) (see, e.g., Weintraub, B, Principles of Radioimmunoassays, seven Training couse one radioactive and Assay technologies, The Endocrine Society, 3 months 1986, incorporated herein by reference). The radioisotope may be detected by means such as the use of a gamma counter or scintillation counter or by autoradiographic methods.

Pharmaceutical composition

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of monoclonal antibodies of the invention, or antigen-binding portions thereof, formulated together with a pharmaceutically acceptable carrier. Such compositions may comprise one or a combination (e.g. two or more different) of the antibodies or immunoconjugates or bispecific molecules of the invention. For example, the pharmaceutical compositions of the invention may contain a combination of antibodies (or immunoconjugates or bispecific molecules) that bind to different epitopes on the target antigen or have complementary activity.

The pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e. in combination with other agents. For example, a combination therapy may include an anti-fucosyl-GM 1 antibody of the invention in combination with at least one other anti-inflammatory agent or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate or bispecific molecule, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable salts. "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, e.g., Berge, s.m. et al (1977) j.pharm.sci.66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as those derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous or nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the sterilization procedures described above or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical can be brought about by the inclusion of absorption delaying agents, for example, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the composition.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable drug can be achieved by incorporating into the composition a delayed absorption agent, such as monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, by 100%, in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be scaled down or up as required by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject being treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifics of the dosage unit forms of the invention are defined and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for use in the treatment of sensitivity in an individual.

For administration of the antibody, the dosage range is about 0.0001 to 100mg/kg, more usually 0.01 to 25mg/kg of the recipient's body weight. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight, or in the range of 1-10mg/kg body weight. An exemplary treatment regimen entails dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. A preferred dosage regimen for the anti-fucosyl-GM 1 antibody of the invention comprises intravenous administration of 1mg/kg body weight or 3mg/kg body weight of the antibody using one of the following dosage regimens: (i) once every 4 weeks for 6 times, then once every 3 months; (ii) once every 3 weeks; (iii) once at 3mg/kg body weight and then 1mg/kg body weight every 3 weeks.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the administered dose of each antibody falls within the ranges. The antibody is usually administered multiple times as necessary. The interval between single administrations can be, for example, weekly, monthly, every three months, or yearly. Intervals can also be irregular, for example, by determining blood levels of antibodies against the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μ g/ml, and in some methods about 25-300 μ g/ml.

Alternatively, the antibody may also be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over an extended period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until progression of the disease is reduced or halted, preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the administration to the patient may be carried out in a prophylactic regime.

The actual dosage level of the active ingredient in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and like factors well known in the medical arts.

The "therapeutically effective dose" of the anti-fucosyl-GM 1 antibody of the invention preferably results in a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of injury or disability due to the affliction of the disease. For example, for treatment of a tumor, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, more preferably by at least about 60%, more preferably by at least about 80%, relative to an untreated subject. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, such a property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, which inhibition can be measured in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a therapeutic compound is capable of reducing tumor size or otherwise alleviating a symptom in a subject. Such amounts can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The compositions of the present invention may be administered by one or more routes of administration using one or more methods well known in the art. It will be appreciated by those skilled in the art that the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injections, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

Alternatively, the antibodies of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration.

The active compounds can be formulated with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or are generally known to those skilled in the art. See, for example, Sustainated and controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The therapeutic compositions can be administered using medical devices well known in the art. For example, in a preferred embodiment, the therapeutic compositions of the present invention can be administered using a needleless hypodermic injection device, such as those described in U.S. Pat. Nos.5,399,163; 5,383,851, respectively; 5,312,335, respectively; 5,064,413, respectively; 4,941,880, respectively; 4,790,824, respectively; or 4,596,556. Examples of known implants and modules that may be used in the present invention include: U.S. patent No.4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent No.4,486,194, which discloses a therapeutic device for transdermal drug delivery; U.S. Pat. No.4,447,233, which discloses a medical infusion pump for delivering a drug at a precise infusion rate; U.S. patent No.4,447,224, which discloses a variable flow implantable infusion device for continuous delivery of a drug; U.S. patent No.4,439,196, which discloses an osmotic drug delivery system having multi-chambered compartments: and U.S. patent No.4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) prevents many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention are able to cross the BBB (if desired), they may be formulated, for example, in liposomes. As for methods of preparing liposomes, see, for example, U.S. Pat. nos. 4,522,811; 5,374,548, respectively; and 5,399,331. Liposomes comprise one or more moieties that can be selectively transported into a particular cell or organ, thereby enhancing targeted drug delivery (see, e.g., v.v. ranade (1989) j.clin.pharmaco 1.29: 685). Examples of targeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al); mannoside (Umezawa et al (1988) biochem. Biophys. Res. Commun.153: 1038); antibodies (P.G.Bloeman et al (1995) FEBS Lett.357: 140; M.Owais et al (1995) Antimicrob.AgentsChemother.39: 180); the surfactant protein A receptor (Briscoe et al (1995) am. J. physiol.1233: 134); p120(Schreier et al (1994) J.biol.chem.269: 9090); see also k.keinanen; m.l. laukkanen (1994) FEBS lett.346: 123; j.j.killion; fidler (1994) immunolmethods 4: 273.

applications and methods of the invention

The antibodies, antibody compositions and methods of the invention have a number of in vitro and in vivo diagnostic and therapeutic applications relating to the diagnosis and treatment of fucosyl-GM 1-mediated diseases. In a preferred embodiment, the antibody of the invention is a human antibody. For example, these molecules can be administered to cells cultured in vitro or ex vivo, or to human subjects, e.g., in vivo, for the treatment, prevention, and diagnosis of various diseases. The term "subject" as used herein includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients having a disease mediated by fucosyl-GM 1 activity or having a disease consistent with fucosyl-GM 1 mediated activity. These methods are particularly suitable for treating human patients suffering from diseases associated with aberrant fucosyl-GM 1 expression or increased fucosyl-GM 1 levels. When the anti-fucosyl-GM 1 antibody is administered together with another drug, the two drugs may be administered sequentially or simultaneously.

Whereas the antibody of the present invention specifically binds to fucosyl-GM 1, the antibody of the present invention can be used to specifically detect the expression of fucosyl-GM 1 on the cell surface, and can be used to purify fucosyl-GM 1 by immunoaffinity purification methods.

The present invention further provides a method of detecting the presence of fucosyl-GM 1 antigen or determining the amount of fucosyl-GM 1 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to fucosyl-GM 1 under conditions that allow the formation of a complex between the antibody, or portion thereof, and fucosyl-GM 1. The formation of complexes is then detected, wherein a difference in complex formation between the sample and the control sample indicates the presence of fucosyl-GM 1 antigen in the sample.

fucosyl-GM 1 is expressed in small cell lung Cancer, but not in normal lung or other tissues (Nilsson et al (1984) Glycoconjugate J1: 43-9; Krug et al, Clin Cancer Res 10: 6094-100). anti-fucosyl-GM 1 antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-fucosyl-GM 1 antibody may also be used with other immunogenic agents, standard cancer therapy or other antibodies.

The anti-fucosyl-GM 1 monoclonal antibody has been shown to mediate strong CDC against fucosyl-GM 1 positive cell lines (Livingston PO et al, (1994) J ClinOncol.12: 1036-44; Brezicka et al, (2000) cancer Immunol Immunother 49: 235-42). Further, it has been reported that fucosyl-GM 1-specific monoclonal antibody-induced complement activation in combination with cytostatic drugs resulted in a synergistic cytotoxic effect against fucosyl-GM 1-expressing cell lines (Brezicka and Einbeigi (2001) Tumour Biol 22: 97-103). Finally, anti-fucosyl-GM 1 monoclonal antibodies have been reported to inhibit the transplantation of fucosyl-GM 1 expressing tumor cells in nude mice (Brezicka et al (1991) Int Jcancer 49: 911-8). These data support the development of fully human monoclonal antibodies against fucosyl-GM 1 as immunotherapeutics for SCLC alone or in combination with chemotherapeutic agents.

Preferred cancers whose growth can be inhibited with the antibodies of the invention include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred cancers that may be treated include lung cancer (including small cell lung cancer and non-small cell lung cancer). Examples of other cancers that may be treated by the methods of the invention include colon cancer (including small bowel cancer), lung cancer, breast cancer, pancreatic cancer, melanoma (e.g., metastatic malignant melanoma), acute myeloid leukemia, kidney cancer, bladder cancer, ovarian cancer and prostate cancer, kidney cancer (including renal cell cancer), glioblastoma, brain tumor, chronic or acute leukemia (including Acute Lymphocytic Leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, burkitt's lymphoma, Anaplastic Large Cell Lymphoma (ALCL), cutaneous T-cell lymphoma, nodular small-cell-cleaved cell lymphoma, Peripheral T cell lymphoma, lunett lymphoma, immunoblastic lymphoma, T cell leukemia/lymphoma (ATLL), central blastic/central cellular (cb/cc) follicular lymphoma, diffuse large cell lymphoma of the B lineage, angioimmunoblastic lymphadenopathy (AILD) -like T cell lymphoma and HIV-associated coelomic lymphoma), embryonal carcinoma, undifferentiated nasopharyngeal carcinoma (e.g., schmink's tumor), castleman's disease, kaposi's sarcoma, multiple myeloma, waldenstrom's macroglobulinemia and other B cell lymphomas, nasopharyngeal carcinoma, bone cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, esophageal cancer, small cell carcinoma, small cell lymphoma, cervical cancer, vaginal cancer, vulval cancer, cervical cancer, cancer of the endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, urinary tract, penile, childhood solid tumors, bladder, kidney or ureter, renal pelvis, Central Nervous System (CNS) tumors, tumor angiogenesis, spinal column tumors, brain stem glioma, pituitary adenoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including asbestos-induced cancers, such as mesothelioma, and combinations of said cancers.

Furthermore, given that fucosyl-GM 1 is expressed on a variety of tumor cells, the human antibodies, antibody compositions, and methods of the invention can be used to treat patients with tumorigenic diseases, such as diseases characterized by the presence of fucosyl-GM 1-expressing tumor cells, including, for example: lung cancer (including small cell lung cancer and non-small cell lung cancer), colon cancer (including small bowel cancer), melanoma (e.g., metastatic malignant melanoma), acute myeloid leukemia, lung cancer, breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and prostate cancer. Examples of other patients with neoplastic disease include patients with: renal cancer (e.g., renal cell carcinoma), glioblastoma, brain tumor, chronic or acute leukemia (including Acute Lymphocytic Leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, burkitt's lymphoma, Anaplastic Large Cell Lymphoma (ALCL), cutaneous T-cell lymphoma, nodular small cleaved cell lymphoma, peripheral T-cell lymphoma, lunett lymphoma, immunoblastic lymphoma, T-cell leukemia/lymphoma (ATLL), central blastic/central (cb/cc) follicular lymphoma, B-lineage diffuse large cell lymphoma, B-lineage lymphoma, c-lymphoblastic lymphoma, c-cell leukemia/lymphoma, c-cell, Angioimmunoblastic lymphadenopathy (AILD) -like T-cell lymphoma and HIV-associated coelomic lymphoma), embryonal carcinoma, undifferentiated nasopharyngeal carcinoma (e.g., Schmingkin's tumor), Karlmann's disease, Kaposi's sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia and other B-cell lymphomas, nasopharyngeal carcinoma, bone carcinoma, skin carcinoma, head and neck carcinoma, cutaneous or intraocular malignant melanoma, uterine carcinoma, rectal carcinoma, anal region carcinoma, gastric carcinoma, testicular carcinoma, uterine carcinoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulval carcinoma, esophageal carcinoma, small intestine carcinoma, cancer of the endocrine system, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urinary tract carcinoma, penile carcinoma, solid tumors of children, bladder carcinoma, renal or ureter carcinoma, renal pelvis carcinoma, tumors of the Central Nervous System (CNS), tumor angiogenesis, spinal tumor, Brain stem glioma, pituitary adenoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers such as mesothelioma), and combinations of said cancers.

Accordingly, in one embodiment, the present invention provides a method of inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of an anti-fucosyl-GM 1 antibody, or an antigen-binding portion thereof. Preferably, the antibody is a human anti-fucosyl-GM 1 antibody (any human anti-fucosyl-GM 1 antibody as described herein). Additionally or alternatively, the antibody may be a chimeric or humanized anti-fucosyl-GM 1 antibody.

In one embodiment, the antibodies (e.g., human monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention can be used to detect the level of fucosyl-GM 1 or a cell containing fucosyl-GM 1 on its membrane surface, which can then be correlated with a particular disease symptom. Alternatively, these antibodies can be used to inhibit or block fucosyl-GM 1 function, which in turn can be correlated with prevention or amelioration of symptoms of a particular disease, thereby allowing fucosyl-GM 1 to act as a mediator of disease. This can be achieved as follows: the experimental and control samples were contacted with anti-fucosyl-GM 1 antibody under conditions that allowed the formation of a complex between the antibody and fucosyl-GM 1. Any complexes formed between the antibody and fucosyl-GM 1 were detected and compared in the experimental samples and controls.

In another embodiment, the binding activity of the antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) of the invention can be initially tested in connection with in vitro therapeutic or diagnostic applications. For example, the compositions of the present invention can be detected using the flow cytometry assays described in the examples below.

The antibodies (e.g., human antibodies, multispecific and bispecific molecules, immunoconjugates and compositions) of the invention have additional applications in the treatment and diagnosis of fucosyl-GM 1-related diseases. For example, human monoclonal antibodies, multispecific or bispecific molecules, and immunoconjugates can be used to elicit one or more of the following biological activities in vivo or in vitro: inhibiting the growth and/or killing a cell expressing fucosyl-GM 1; mediating phagocytosis or ADCC of fucosyl-GM 1 expressing cells in the presence of human effector cells; or blocking the binding of fucosyl-GM 1 ligand to fucosyl-GM 1.

In a particular embodiment, antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat, prevent or diagnose a variety of fucosyl-GM 1-associated diseases. Examples of fucosyl-GM 1-associated diseases include, inter alia, lung cancer (including small cell lung cancer and non-small cell lung cancer).

Suitable routes for administering the antibody compositions of the invention (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by one of skill in the art. For example, the antibody composition can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, the human anti-fucosyl-GM 1 antibody of the invention may be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody may be linked to the therapeutic agent (as an immune complex) or may be administered separately from the therapeutic agent. For the latter (separate administration), the antibody may be administered before, after or simultaneously with the therapeutic agent, or may be co-administered with other known therapies such as anti-cancer therapies, e.g., radiation therapy. These therapeutic agents include, inter alia: antineoplastic agents, such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide, hydroxyurea, are themselves effective only at levels that are toxic or sub-toxic to the patient. Cisplatin was administered intravenously at a dose of 100 mg/dose 1 time every 4 weeks, and doxorubicin at a dose of 60-75mg/ml 1 time every 21 days. Co-administration of the human anti-fucosyl-GM 1 antibody or antigen-binding fragment thereof of the invention with a chemotherapeutic agent provides two anti-cancer agents that act through different mechanisms that produce cytotoxic effects on human tumor cells. Such co-administration can solve problems caused by the development of drug resistance or antigenic changes in tumor cells that would render them non-reactive to antibodies.

In one embodiment, immunoconjugates of the invention can be used to target a compound (e.g., a therapeutic agent, a label, a cytotoxin, a radiotoxin, an immunosuppressive agent, etc.) to a cell having a fucosyl-GM 1 cell surface receptor by linking the compound to an antibody. For example, an anti-fucosyl-GM 1 antibody may be conjugated to any one of the toxin compounds described in U.S. patent nos. 6,281,354 and 6,548,530, U.S. patent publication nos.20030050331, 20030064984, 20030073852 and 20040087497, or WO03/022806 (which are incorporated herein by reference in their entirety). Thus, the invention also provides methods for localizing cells expressing fucosyl-GM 1 ex vivo or in vivo (e.g., using a detectable label such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). Alternatively, immunoconjugates may also be used to kill cells having fucosyl-GM 1 cell surface receptors by targeting a cytotoxin or radiotoxin to fucosyl-GM 1.

Target-specific effector cells, such as effector cells linked to compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules), can also be used as therapeutic agents. The effector cells for targeting may be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other cells bearing IgG or IgA receptors. If desired, effector cells may be obtained from the subject to be treated. The target-specific effector cells may be administered as a suspension of the cells in a physiologically acceptable solution. The number of cells administered may be 10⁸-10⁹Of the order of magnitude, but differs according to the therapeutic purpose. Typically, this amount is sufficient to obtain localization at the target cell, e.g. at a tumor cell expressing fucosyl-GM 1, and killing of the cell is achieved by e.g. phagocytosis. The route of administration may also vary.

Treatment with target-specific effector cells may be performed in conjunction with other techniques for clearing target cells. For example, anti-tumor treatments using the compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules) and/or effector cells bearing these compositions can be used in conjunction with chemotherapy. In addition, combination immunotherapy can be applied to direct two distinct cytotoxic effector populations to tumor cell rejection. For example, an anti-fucosyl-GM 1 antibody linked to anti-Fc- γ RI or anti-CD 3 may be used with an IgG or IgA receptor specific binding agent.

The bispecific and multispecific molecules of the invention may also be used to modulate Fc γ R or Fc γ R levels on effector cells, for example by capping (trapping) and clearance of receptors on the cell surface. Mixtures of anti-Fc receptor antibodies can also be used for this purpose.

Compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules, and immunoconjugates) having a complement binding site (e.g., a complement-binding portion from IgG1, -2 or-3 or IgM) can also be used in the presence of complement. In one embodiment, ex vivo treatment of a cell population containing target cells with a binding agent of the invention and suitable effector cells can be achieved by addition of complement or complement-containing serum. Binding of complement proteins can improve phagocytosis of target cells coated with the binding agents of the invention. In another embodiment, target cells coated with the compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules) are also capable of being lysed by complement. In another embodiment, the compositions of the invention do not activate complement.

Compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules, and immunoconjugates) can also be administered with complement. Thus, compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement are also within the scope of the invention. These compositions are advantageous because complement is in close proximity to human antibodies, multispecific or bispecific molecules. Alternatively, the human antibody, multispecific or bispecific molecule and complement or serum of the invention can also be administered separately.

Thus, a patient treated with an antibody composition of the invention (either prior to, concurrently with, or subsequent to administration of a human antibody of the invention) may be additionally administered another therapeutic agent, such as a cytotoxic or radiotoxic agent, which may enhance or augment the therapeutic effect of the human antibody.

In other embodiments, the subject may additionally be treated with a drug that modulates (e.g., enhances or inhibits) the expression or activity of Fc γ or Fc γ receptors, e.g., a cytokine. Preferred cytokines for administration during treatment with multispecific molecules include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and Tumor Necrosis Factor (TNF).

The compositions of the invention (e.g., human antibodies, multispecific and bispecific molecules) can also be used to target, e.g., label, cells expressing Fc γ R or fucosyl-GM 1. For such applications, the binding agent may be linked to a molecule that is capable of being detected. Accordingly, the present invention provides methods for ex vivo or in vitro localization of cells expressing Fc receptors such as Fc γ R or fucosyl-GM 1. The detectable label may be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

Also included within the scope of the invention are kits comprising an antibody composition of the invention (e.g., a human antibody, a multispecific or bispecific molecule, or an immunoconjugate) and instructions for use. The kit may further comprise at least one additional agent such as an immunosuppressive, cytotoxic or radiotoxic agent or one or more additional human antibodies of the invention (e.g., a human antibody with complementary activity that binds to a different epitope on the fucosyl-GM 1 antigen than the first human antibody). The kit will generally include a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded material affixed to or otherwise provided with the kit.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited in this application are incorporated herein by reference.

Examples

Example 1: generation of human monoclonal antibodies to fucosyl-GM 1

Antigens

Immunization protocols used fucosyl-GM 1(Northwest Biotherpeutics, Inc.) adsorbed by Salmonella Minnesota as an antigen.

Transgenic HuMab and KM mice ^TM

Fully human monoclonal antibodies against fucosyl-GM 1 were prepared with HCo7, HCo12, HCo7+ HCo12 strains of HuMab transgenic mice expressing human antibody genes and KM strains of transgenic transchromosomal mice. In each of these mouse strains, a strain has been described by Chen et al (1993) EMBO J.12: 811-820 the endogenous mouse kappa light chain gene has been homozygously disrupted and the endogenous mouse heavy chain gene has been homozygously disrupted as described in example 1 of PCT publication WO 01/09187. These mouse strains all carry a strain such as Fishwild et al (1996) Nature Biotechnology 14: 845 ℃ 851 the human kappa light chain transgene KCo 5. The HCo7 strain carries the HCo7 human heavy chain transgene as described in U.S. patent nos.5,770,429, 5,545,806, 5,625,825 and 5,545,807. The HCo12 strain carries the HCo12 human heavy chain transgene as described in example 2 of WO01/09187 or example 2 of WO 01/14424. The HCo7+ HCo12 strain carries the HCo7 and HCo12 heavy chain transgenes. The KM strain contains SC20 transchromosome as described in PCT publication WO 02/43478. All of these strains are referred to herein as HuMAb mice.

Immunization with HuMab and KM：

To generate fully human monoclonal antibodies against fucosyl-GM 1, HuMab and KM mice were immunized with fucosyl-GM 1(Northwest Biotherpeutics, Inc.) adsorbed onto the surface of acid-treated Salmonella minnesota by lyophilization^TM. General immunization protocols for HuMab mice are described in Lonberg, n. et al (1994) Nature368 (6474): 856-859; fishwild, D. et al (1996) Nature Biotechnology 14: 845, 851 and PCT publication WO 98/24884. Mice were 6-16 weeks old at the first infusion of antigen.

Transgenic mice were immunized twice Intraperitoneally (IP) or subcutaneously (Sc) with antigen in complete freund's adjuvant, and then intraperitoneally with antigen in incomplete freund's adjuvant for 2-4 weeks (up to a total of 8 immunizations). The immune response was monitored by ELISA (described below) on serum obtained from retroorbital bleeds. Fusion was performed with mice with sufficient titers of anti-fucosyl-GM 1 human immunoglobulin. Mice were boosted intravenously with antigen, sacrificed 3 and 2 days later, and spleens were removed.

HuMab or KM mice that produce anti-fucosyl-GM 1 antibodies ^TM Selection of

To select HuMab or KM mice that produce antibodies that bind to fucosyl-GM 1^TMSera from immunized mice were tested by ELISA as described by Fishwild, D. et al (1996). Briefly, native fucosyl-GM 1 purified from a fucosyltransferase transfected cell line (Northwest Biothereutics, Inc.) or from bovine brain (Matreya, Inc.) was dissolved at 1mg/ml in methanol and passively adsorbed on polypropylene microtiter plates at 50. mu.l/well by air-drying at room temperature for 1-2 hours. Similarly, plates coated with a relevant control antigen such as GM1 were prepared as counter-screen plates (counter screen) for cross-reactive antibodies. Assay plates were then blocked with 250. mu.l/well of 1% ovalbumin in PBS for 1 hour at room temperature. Plasma dilutions from fucosyl-GM 1 immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS and then incubated with goat anti-human IgG Fc or goat anti-human IgM Fc polyclonal antibodies conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the assay plate was developed with TMB substrate and analyzed with a spectrophotometer at OD450 nm.

The fusion was performed with mice showing the highest anti-fucosyl-GM 1 antibody titers. Fusions were performed as described below and hybridoma supernatants were tested for anti-fucosyl-GM 1 activity by ELISA.

Generation of hybridomas producing anti-fucosyl-GM 1 human monoclonal antibodies

FromMouse and/or KM mouse^TMSplenocytes were isolated and fused with a mouse myeloma cell line using a Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences, inc., Glen Burnie, MD) using standard PEG-based procedures or electric field-based electrofusion. Then based on antigen specificityProduction of antibody the resulting hybridomas were screened.

Single cell suspensions of splenic lymphocytes from immunized mice were fused with 1/4 numbers of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC CRL1580) or SP2/0 non-secreting mouse myeloma cells (ATCC CRL1581) using 50% PEG (Sigma). The cells were cultured at approximately 1 × 10⁵The density of/well was plated on flat-bottomed microtiter plates and incubated for approximately two weeks in selective medium containing 10% fetal bovine serum, 10% P388D1(ATCC, CRL TIB-63) conditioned medium, 3-5% origen (IGEN) in medium with HT replaced HAT in DMEM (Mediatech, CRL10013 containing high concentrations of glucose, L-glutamine and sodium pyruvate) plus 5mM HEPES, 0.055mM β -mercaptoethanol, 50mg/ml gentamicin and 1 × HAT (Sigma; CRL P-7185).

Hybridomas having specific binding activity in an ELISA assay screen were further tested for specific binding to fucosyl-GM 1 adsorbed onto mammalian cells by FACS (described below). Hybridomas showing the highest specific binding according to ELISA and FACS were subcloned at least twice by limiting dilution. The resulting stable subclones are then cultured in vitro to produce small amounts of monoclonal antibodies in tissue culture medium. ELISA screening was repeated to verify subclone activity. The subclones that showed the highest activity in the ELISA were scaled up to produce enough conditioned medium (typically 1L) for purification of monoclonal anti-fucosyl-GM 1 for further characterization.

Hybridoma clones 5B1, 5B1a, 7D4, 7E4, 13B8, 13B8a, and 18D5 were selected for further analysis.

Example 2: structural characterization of human monoclonal antibodies 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5

The cDNA sequences encoding the heavy and light chain variable regions of the 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 monoclonal antibodies were obtained from 5B1, 5B1a, 7D4, 7E4, 13B8 and 18D5 hybridomas, respectively, using standard PCR techniques and sequenced using standard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variable region of 5B1 are shown in fig. 1A and SEQ ID NO: 49 and 1.

The nucleotide and amino acid sequences of the light chain variable region of 5B1 are shown in fig. 1B and SEQ ID NO: 55 and 7.

Comparison of the 5B1 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 5B1 heavy chain employs a VH segment from human germline VH3-48, a D segment from human germline 1-1, and a JH segment from human germline JH 6B. An alignment of the 5B1VH sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 5B1VH sequence using the Kabat CDR region assay system outlined the heavy chain CDR1, CDR2 and CDR3 regions as shown in fig. 1A and 7 and SEQ ID NO: 13. 19 and 25.

Comparison of the 5B1 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 5B1 light chain utilized VL segments from human germline VK L15 and JK segments from human germline JK 4. An alignment of the 5B1VL sequence with the germline VK L15 sequence is shown in fig. 8. Further analysis of the 5B1VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 1B and 8 and SEQ ID NO: 31. 37 and 43.

The nucleotide and amino acid sequences of the heavy chain variable region of 5B1a are shown in fig. 2A and SEQ ID NO: 50 and 2.

The nucleotide and amino acid sequences of the light chain variable region of 5B1a are shown in fig. 2B and SEQ ID NOs: 56 and 8.

Comparison of the 5B1a heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 5B1a heavy chain employs a VH segment from human germline VH3-48 and a D segment from human germline 1-1 and a JH segment from human germline JH 6B. An alignment of the 5B1 ahv sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 5B1a vh sequence using the Kabat CDR region assay system outlined the heavy chain CDR1, CDR2, and CDR3 regions as shown in fig. 2A and 7 and SEQ ID NO: 14. 20 and 26.

Comparison of the 5B1a light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 5B1a light chain utilized a VL segment from human germline VK L15 and a JK segment from human germline JK 4. An alignment of the 5B1a VL sequence to the germline VKL15 sequence is shown in fig. 8. Further analysis of the 5B1a VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 2B and 8 and SEQ ID NO: 32. 38 and 44.

The nucleotide and amino acid sequences of the heavy chain variable region of 7D4 are shown in fig. 3A and SEQ ID NO: 51 and 3.

The nucleotide and amino acid sequences of the light chain variable region of 7D4 are shown in fig. 3B and SEQ ID NO: 57 and 9.

Comparison of the 7D4 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 7D4 heavy chain employs a VH segment from human germline VH3-48, a D segment from human germline 1-1, and a JH segment from human germline JH6 b. An alignment of the 7D4VH sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 7D4VH sequence using the Kabat CDR region assay system outlined the heavy chain CDR1, CDR2 and CDR3 regions as shown in fig. 3A and 7 and SEQ ID NO: 15. 21 and 27.

Comparison of the 7D4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 7D4 light chain utilized VL segments from human germline VK L15 and JK segments from human germline JK 4. An alignment of the 7D4VL sequence with the germline VK L15 sequence is shown in fig. 8. Further analysis of the 7D4VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 3B and 8 and SEQ ID NO: 33. 39 and 45.

The nucleotide and amino acid sequences of the heavy chain variable region of 7E4 are shown in fig. 4A and SEQ ID NO: 52 and 4.

The nucleotide and amino acid sequences of the light chain variable region of 7E4 are shown in fig. 4B and SEQ ID NO: 58 and 9.

Comparison of the 7E4 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 7E4 heavy chain employs a VH segment from human germline VH3-48, a D segment from human germline 1-1, and a JH segment from human germline JH6 b. An alignment of the 7E4VH sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 7E4VH sequence using the Kabat CDR region assay system outlined the heavy chain CDR1, CDR2 and CDR3 regions as shown in fig. 4A and 7 and SEQ ID NO: 16. 22 and 28.

Comparison of the 7E4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 7E4 light chain utilized VL segments from human germline VK L15 and JK segments from human germline JK 4. An alignment of the 7E4VL sequence with the germline VK L15 sequence is shown in fig. 8. Further analysis of the 7E4VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 4B and 8 and SEQ ID NO: 34. 40 and 46.

The nucleotide and amino acid sequences of the heavy chain variable region of 13B8 are shown in fig. 5A and SEQ ID NO: 53 and 5.

The nucleotide and amino acid sequences of the light chain variable region of 13B8 are shown in fig. 5B and SEQ ID NO: 59 and 11.

Comparison of the 13B8 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 13B8 heavy chain employs a VH segment from human germline VH3-48, a D segment from human germline 1-1, and a JH segment from human germline JH 6B. An alignment of the 13B8VH sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 13B8VH sequence using the Kabat CDR region determination system outlined the heavy chain CDR1, CDR2 and CDR3 regions as shown in fig. 5A and 7 and SEQ id no: 11. 17 and 23.

Comparison of the 13B8 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 13B8 light chain utilized VL segments from human germline VK L15 and JK segments from human germline JK 4. An alignment of the 13B8VL sequence with the germline VKL15 sequence is shown in fig. 8. Further analysis of the 13B8VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 5B and 8 and SEQ ID NO: 35. 41 and 47.

The nucleotide and amino acid sequences of the heavy chain variable region of 3C4 are shown in fig. 6A and SEQ ID NO: 54 and 63.

The nucleotide and amino acid sequences of the light chain variable region of 3C4 are shown in fig. 6B and SEQ ID NO: 60 and 64.

Comparison of the 18D5 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 18D5 heavy chain employs a VH segment from human germline VH3-48, a D segment from human germline 1-1, and a JH segment from human germline JH6 b. An alignment of the 18D5VH sequence with germline VH3-48 sequences is shown in fig. 7. Further analysis of the 18D5VH sequence using the Kabat CDR region assay system outlined the heavy chain CDR1, CDR2 and CDR3 regions as shown in fig. 7 and SEQ ID NO: 18. 24 and 30.

Comparison of the 18D5 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 18D5 light chain utilized VL segments from human germline VK L15 and JK segments from human germline JK 4. An alignment of the 18D5VL sequence with the germline VKL15 sequence is shown in fig. 8. Further analysis of the 18D5VL sequence using the Kabat CDR region determination system outlined the light chain CDR1, CDR2 and CDR3 regions as shown in fig. 8 and SEQ ID NO: 36. 42 and 48.

Example 3: preparation of cells doped with fucosyl-GM 1

Cells containing fucosyl-GM 1 embedded in the plasma membrane were prepared for use in binding assays. Purified fucosyl-GM 1 was dissolved in a 1: 1 mixture of chloroform: methanol in a glass tube and evaporated to dryness under nitrogen. Ca-free addition to dry fucosyl-GM 1Or Mg in PBS to give a concentration of 200ug/ml of antigen, generating an emulsion by vortexing for 5 minutes and then sonicating for 5 minutes, 2 × 10⁶Density of cells/ml suspensions of target cells (typically HEK293 (human kidney, ATCC # CRL-1573) or Daudi (human Burkitt's lymphoma, ATCC # CCL-213)) were prepared in PBS. Equal volumes of fucosyl-GM 1 emulsion and cell suspension were mixed to 100ug antigen/10⁶Final concentration of cells/ml cells were incubated with antigen at 37 ℃ for 15 minutes to allow fucosyl-GM 1 to insert into the membrane, then incubated at room temperature for 30 minutes with occasional mixing throughout the process, cells were centrifuged at 1000rpm for 10 minutes, the supernatant was discarded, and "spiked" cells were incubated at 4 × 10⁶The cells/ml were resuspended in FACS buffer (PBS without Ca or Mg + 1% human serum + 2% PBS +2mM EDTA). Similarly, control cells were prepared that were either spiked with the relevant antigen, such as GM1, or that did not contain the antigen.

Example 4: characterization of binding specificity and binding kinetics of anti-fucosyl-GM 1 human monoclonal antibodies

Binding specificity determined by ELISA

Specific binding of purified monoclonal human anti-fucosyl-GM 1 antibody to purified fucosyl-GM 1 was detected by ELISA using recombinant antigen and spiked cells. All steps were performed on ice. Conditioned media from ELISA positive hybridoma cultures were mixed with antigen or cells in "V" plates and incubated for 1 hour. Cells were washed and bound antibody was detected with HRP conjugated mouse anti-human IgG Fc secondary antibody. After washing, the plate is developed with a chromogenic substrate, pelleted by centrifugation, and the supernatant transferred to a flat bottom microtiter assay plate for analysis using a spectrophotometer. The results are shown in fig. 9 (antigen ELISA) and fig. 10 (whole cell ELISA). anti-fucosyl-GM 1 antibody was shown to bind specifically to fucosyl-GM 1.

Binding specificity determined by flow cytometry

The specificity of the fucosyl-GM 1 human monoclonal antibodies was tested by flow cytometry using naturally expressed fucosyl-GM 1 positive cell lines such as H-4-II-E (rat hepatoma ATCC # CRL-1548) or DMS-79 (human SCLC ATCC # CRL-2049). All staining steps were performed on ice. Binding of anti-fucosyl-GM 1 human monoclonal antibody was assessed as follows: transfected cells were incubated with anti-fucosyl-GM 1 human monoclonal antibody at a concentration of 10. mu.g/ml. Cells were washed and binding was detected with FITC-labeled anti-human IgG antibody. Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson, san Jose, Calif.). The results are shown in FIG. 11. The anti-fucosyl-GM 1 human monoclonal antibody binds to H-4-II-E and DMS-79 cell lines. This data demonstrates the specificity of human monoclonal antibodies to fucosyl-GM 1 for fucosyl-GM 1.

Example 5: internalization of anti-fucosyl-GM 1 monoclonal antibodies

The ability of anti-fucosyl-GM 1HuMAb to internalize into fucosyl-GM 1 expressing cell lines was examined using the Hum-Zap internalization assay. The Hum-Zap assay detects internalization of a first human antibody by binding of a second antibody that has affinity for human IgG conjugated to the toxin saporin (saporin).

Cell lines expressing fucosyl-GM 1 (H-4-II-E or DMS-79) were suspended in medium and added to microtiter cell culture plates at a density of 7500 cells/well. Serial dilutions of test antibody and isotype control were prepared in culture medium and conjugated to a 2: 1 molar excess of saporin-conjugated secondary antibody (Hum-Zap) on ice^TMAdvanced Targeting Systems, San Diego, CA, IT-22-25) for 1 hour. The antibody + saporin conjugate mixture was then mixed with the cells and incubated at 37 ℃ for 48-72 hours. MTS (Promega) was added to test cell viability. After an additional 4 hours of incubation, the OD was read and the absorbance was inversely proportional to the internalization. The results are shown in fig. 12. This data demonstrates that human anti-fucosyl-GM 1 antibody can be internalized into cell lines expressing fucosyl-GM 1.

Example 6: complement dependent cytotoxicity effects of anti-fucosyl-GM 1 antibodies

Target cells, i.e. "doped" or naturally expressed cell lines, are suspended in CDC buffer (RPMI1640) to a density of 10⁶and/mL. Human Complement (HC) was diluted 1: 3 in cell line growth medium. Serial dilutions of the test antibody and isotype control were prepared. Cells, complement and antibody were mixed in equal volumes in microtiter assay plates and incubated at 37 ℃ for 2 hours. Alamar blue was added to each well and the plates were incubated at 37 ℃ for a further 21 hours. The plates were read using a fluorescence plate reader using 530nm absorption/590 nm emission spectra, with cell viability proportional to fluorescence units. The results are shown in fig. 13. Human and mouse control antibodies exhibit strong, dose-dependent CDC activity against DMS79 and H-4-II-E cells. Isotype control antibodies showed no significant cytotoxicity. CDC activity was negligible on fucosyl-GM 1 negative cell lines such as Ramos and ARH 77.

Example 7: evaluation of ADCC Activity of anti-fucosyl-GM 1 antibody

In this example, the ability of an anti-fucosyl-GM 1 monoclonal antibody to kill fucosyl-GM 1 expressing cell lines by Antibody Dependent Cellular Cytotoxicity (ADCC) in the presence of effector cells was examined using a fluorescence cytotoxicity assay.

ADCC Activity was measured using the Delfia System (Perkin-Elmer) briefly, effector cells were cultured from human Peripheral Blood Mononuclear Cells (PBMC) by overnight stimulation with 200u/ml IL-2 and resuspended at 2 × 10⁷And/ml. Target cells were diluted to 10⁶Perml, loaded with fluorescence enhancing ligand (BATDA) by incubation for 20 min and diluted to 2 × 10⁷Cells/ml. Effector and target cells were mixed in a 100: 1 ratio in microtiter assay plates and mixed with serial dilutions of test antibody and isotype control. The plates were incubated at 37 ℃ for 1 hour, the cells were pelleted by centrifugation,mu.l of the supernatant was removed and mixed with a Eu solution (Perkin-Elmer). The fluorescence emitted by the released ligand mixed with Eu is measured with a Fusion plate reader (Perkin-Elmer) and is proportional to the cell lysis. Assay wells containing effector cells without antibody and assay wells containing detergent control for background lysis and complete lysis, respectively, allow calculation of antibody-specific lysis. The results are shown in fig. 14. This data demonstrates that anti-fucosyl-GM 1 antibodies are cytotoxic to cells expressing fucosyl-GM 1 on the cell surface.

Example 8: immunohistochemical study of Lung cancer

The ability of anti-fucosyl-GM 1HuMAb 7E4 to recognize fucosyl-GM 1 was examined by immunohistochemical studies using clinical biopsies of Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC).

For immunohistochemical studies, 5mm frozen sections were prepared from clinical biopsies of Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC) (Ardais Inc, USA). After drying for 30 minutes, the sections were fixed with methanol (10 minutes at room temperature) and air dried for 5 minutes. Slides were rinsed with PBS and then pre-incubated for 20 minutes with 10% normal goat serum in PBS, followed by incubation with 10mg/ml biotinylated 7E4 in PBS containing 10% normal goat serum for 30 minutes at room temperature. Slides were washed three times with PBS and then incubated with streptavidin-FITC (DAKO) for 30 minutes at room temperature. The slides were washed with PBS and incubated with goat anti-FITC HRP conjugate (DAKO) for 30 min at room temperature. Slides were washed three times with PBS. Diaminobenzidine (Sigma) was used as HRP substrate, resulting in brown staining of 7E 4-bound positive tissues. After washing with distilled water, the slides were counterstained with hematoxylin for 1 minute to reveal the tissue structure. The slides were then washed in running distilled water for 10 seconds and fixed in a glycerol gel (DAKO). Immunohistochemical staining of clinical biopsy tissue showed positive staining for both SCLC and NSCLC sections. Only malignant cells were positive in each case, and adjacent normal lung tissue was not stained. Total distribution ratio of lung cancer was 5/13 test samples (SCLC of 2/6 and NSCLC of 3/7). Immunohistochemistry results for 7E4 binding to normal lung tissue were negative.

Example 9: production of nonfucosylated humabs

Antibodies with reduced numbers of fucosyl residues have been shown to improve ADCC ability of the antibody. In this example, an anti-fucosyl-GM 1HuMAb lacking fucosyl residues was produced.

The CHO cell line Ms704-PF (Biowa, inc., Princeton, NJ) lacking the fucosyltransferase gene FUT8 was electroporated with vectors expressing the anti-fucosyl-GM 1HuMAb heavy and light chains. Drug resistant clones were screened by growth in Ex-Cell 325-PF CHO medium (JRHBOSciences, Lenexa, KS) containing 6mM L-glutamine and 500. mu.g/ml G418(Invitrogen, Carlsbad, Calif.). Clones were screened for IgG expression by standard ELISA assays.

Example 10: in vivo potency of anti-fucosyl-GM 1 human monoclonal antibodies

Mixing DMS79 small cell lung cancer cell (fucosyl-GM 1)⁺) Subcutaneous implantation into male SCID mice (5 × 10)⁶Cells/mouse) for a time sufficient to allow tumor formation (about 8 days). Tumor measurements were made on day 8 post-implantation and were based on the mean tumor volume (about 200 mm)³) The mice were randomly divided into 6 groups of 8 mice each for subsequent antibody treatment. On days 8, 11, 15, 18 and 22 post-implantation, mice were injected intraperitoneally (i.p.) according to the following groups: (A) PBS (vehicle control); (B) 30mg/kg human IgG1 per mouse (isotype control); (C) anti-fucosyl-GM 1 monoclonal antibody 5B1 at 10mg/kg per mouse; (D) anti-fucosyl-GM 1 monoclonal antibody 5B1 at 30mg/kg per mouse; (E) anti-fucosyl-GM 1 monoclonal antibody 7E4 at 10mg/kg per mouse; (F) anti-fucosyl-GM 1 monoclonal antibody 7E4 at 30mg/kg per mouse. The monoclonal antibody compositions used in these experiments had low levels of endotoxicityTumor was measured in three dimensions (height × width × length) using an electronically accurate caliper and tumor volume was calculated, tumor was measured twice a week throughout the experiment (61 days), when tumor reached the indicated tumor endpoint (a specific tumor volume such as 1500 mm)³And/or when the mouse shows signs of discomfort or weight loss of more than about 15%).

To examine whether the anti-fucosyl-GM 1 monoclonal antibody delayed tumor growth, tumors were monitored to reach 1000mm³Days in volume. Both the 7E4 and 5B1 anti-fucosyl-GM 1 monoclonal antibodies significantly delayed tumor growth compared to vehicle and isotype controls (see table 1). Antibody potency appears to be dose dependent, with the 30mg/kg treatment group showing a greater response in each case. Furthermore, the tumor volume in mice treated with 30mg/kg of the 7E4 antibody never reached 1000mm³Mean tumor volume at the end of the study on day 61 was 600mm³。

Table 1.

Treatment of	Tumor volume of 1000mm³Days of the day
		PBS (vehicle control)	34
h-IgG1 (isotype control)	32
		anti-5B 1(10mg/kg)	56
anti-5B 1 (3)0mg/kg)	60
		anti-7E 4(10mg/kg)	57
anti-7E 4(30mg/kg)	＞61

To test whether DMS79 cells continued to express fucosyl-GM 1 in vivo, monoclonal antibodies 7E4 and 5B1 were analyzed for binding to DMS79 cells by FACS before implantation and after recovery of DMS79 cells from untreated tumors. The bound antibodies before and after implantation of DMS79 cells demonstrated maintenance of fucosyl-GM 1 expression levels in vivo (fig. 11C).

Figure 15A shows that all control mice (groups a and B), except one, reached the tumor endpoint by day 61. FIG. 15B shows that two mice in the group treated with 10mg/kg anti-fucosyl-GM 15B 1 antibody (group C) reached a tumor endpoint, and that six mice had a tumor volume of about 600mm³To 1000mm³Whereas 8 mice in the group treated with 30mg/kg anti-fucosyl-GM 15B 1 antibody (group D) did not reach the tumor end point on day 61 (volume 1000 mm)³Or smaller). FIG. 15C shows that 8 mice in the group treated with 10mg/kg anti-fucosyl-GM 1 monoclonal antibody 7E4 (group E) did not reach a tumor endpoint by day 61 (volume approximately 1200 mm)³Or smaller, one mouse without tumor). FIG. 15C also shows that 8 mice in the group treated with 30mg/kg anti-fucosyl-GM 1 monoclonal antibody 7E4 (group F) did not reach a tumor endpoint by day 61 (volume of about 800 mm)³Or smaller, two mice had no tumor). Fig. 16 shows (a) mean and (B) median tumor volumes measured on day 61. Antibody efficacy appeared to be dose-dependent, with the 30mg/kg treated group showing a greater response than the control group in each case.

Table 2.

This study showed that anti-fucosyl-GM 1 antibody treatment acted in a dose-dependent manner in murine tumor models and had a significantly stronger effect on tumor growth than vehicle and isotype controls. anti-fucosyl-GM 1 antibody treatment also did not result in weight loss in mice or any other significant side effects, indicating that these antibodies were safe and well tolerated (figure 17). Indeed, the anti-fucosyl-GM 1 antibody showed a percentage inhibition of tumor growth (TGI%) ranging from 81% to 90% at day 32 (table 2). In addition, treatment with antibody 7E4 at a dose of 30mg/kg resulted in 25% of mice being tumor-free.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Accordingly, the disclosure is to be limited only by the following claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

SEQ ID NO：	Sequence of	SEQ ID NO：	Sequence of
				1	VH amino acid 5B1	32	VK CDR1 amino acid 5B1a
2	VH amino acid 5B1a	33	VK CDR1 amino acid 7D4
				3	VH amino acid 7D4	34	VK CDR1 amino acid 7E4
4	VH amino acid 7E4	35	VK CDR1 amino acid 13B8
				5	VH amino acid 13B8	36	VK CDR1 amino acid 18D5
6	VH amino acid 18D5	37	VK CDR2 amino acid 5B1
				7	VK amino acid 5B1	38	VK CDR2 amino acid 5B1a
8	VK amino acid 5B1a	39	VK CDR2 amino acid 7D4
				9	VK amino acid 7D4	40	VK CDR2 amino acid 7E4
10	VK amino acid 7E4	41	VK CDR2 amino acid 13B8
				11	VK amino acid 13B8	42	VK CDR2 amino acid 18D5
12	VK amino acid 18D5	43	VK CDR3 amino acid 5B1
				13	VH CDR1 amino acid 5B1	44	VK CDR3 amino acid 5B1a
14	VH CDR1 amino acid 5B1a	45	VK CDR3 amino acid 7D4
				15	VH CDR1 amino acid 7D4	46	VK CDR3 amino acid 7E4
16	VH CDR1 amino acid 7E4	47	VK CDR3 amino acid 13B8
				17	VH CDR1 amino acid 13B8	48	VK CDR3 amino acid 18D5
18	VH CDR1 amino acid 18D5	49	VH nucleotide 5B1
				19	VH CDR2 amino acid 5B1	50	VH nucleotide 5B1a
20	VH CDR2 amino acid 5B1a	51	VH nucleotide 7D4
				21	VH CDR2 amino acid 7D4	52	VH nucleotide 7E4
22	VH CDR2 amino acid 7E4	53	VH nucleotide 13B8
				23	VH CDR2 amino acid 13B8	54	VH nucleotide 3C4
24	VH CDR2 amino acid 18D5	55	VK nucleotide 5B1
				25	VH CDR3 amino acid 5B1	56	VK nucleotide 5B1a
26	VH CDR3 amino acid 5B1a	57	VK nucleotide 7D4
				27	VH CDR3 amino acid 7D4	58	VK nucleotide 7E4
28	VH CDR3. amino acid 7E4	59	VK nucleotide 13B8
				29	VH CDR3 amino acid 13B8	60	VK nucleotide 3C4
30	VH CDR3 amino acid 18D5	61	VH3-48 germline amino acids
				31	VK CDR1 amino acid 5B1	62	VK L15 amino acid series

Claims

1. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 13, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 19, heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: 25, a heavy chain variable region CDR 3;

(d) as shown in SEQ ID NO: 31, a light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 37, a light chain variable region CDR 2; and

(f) as shown in SEQ ID NO: 43, CDR3 of the light chain variable region.

2. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 14, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 20, a heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: 26, a heavy chain variable region CDR 3;

(d) as shown in SEQ ID NO: 32, light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 38, a light chain variable region CDR 2; and

(f) as shown in SEQ ID NO: 44, CDR3 of the light chain variable region shown.

3. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 15, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 21, heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: a heavy chain variable region CDR3 of 27;

(d) as shown in SEQ ID NO: 33, a light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 39, CDR2 of the light chain variable region shown in seq id no; and

(f) as shown in SEQ ID NO: 45, CDR3 of the light chain variable region.

4. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 16, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 22, a heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: 28, a heavy chain variable region CDR 3;

(d) as shown in SEQ ID NO: 34, light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 40, CDR 2; and

(f) as shown in SEQ ID NO: 46, and a light chain variable region CDR3.

5. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 17, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 23, heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: 29, a heavy chain variable region CDR 3;

(d) as shown in SEQ ID NO: 35, a light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 41, CDR2 of the light chain variable region; and

(f) as shown in SEQ ID NO: 47, and light chain variable region CDR3.

6. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 18, a heavy chain variable region CDR 1;

(b) as shown in SEQ ID NO: 24, a heavy chain variable region CDR 2;

(c) as shown in SEQ ID NO: 30, a heavy chain variable region CDR 3;

(d) as shown in SEQ ID NO: 36, light chain variable region CDR 1;

(e) as shown in SEQ ID NO: 42, light chain variable region CDR 2; and

(f) as shown in SEQ ID NO: 48, CDR3 of the light chain variable region.

7. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 1, or a light chain variable region of the amino acid sequence set forth in seq id no; and

(b) as shown in SEQ ID NO: 7, or a light chain variable region of the amino acid sequence shown in seq id no.

8. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 2; and

(b) as shown in SEQ ID NO: 8 in a light chain variable region of the amino acid sequence shown in seq id No. 8.

9. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 3; and

(b) as shown in SEQ ID NO: 9, or a light chain variable region of the amino acid sequence shown in seq id no.

10. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 4; and

(b) as shown in SEQ ID NO: 10, or a light chain variable region of the amino acid sequence shown in seq id No. 10.

11. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 5; and

(b) as shown in SEQ ID NO: 11, or a light chain variable region of the amino acid sequence shown in seq id No. 11.

12. An isolated human monoclonal antibody, or antigen-binding portion thereof, comprising:

(a) as shown in SEQ ID NO: 6; and

(b) as shown in SEQ ID NO: 12.

13. The antibody, or antigen binding portion thereof, of any one of claims 1-12, which is nonfucosylated.

14. A composition comprising the antibody, or antigen-binding portion thereof, of any one of claims 1-13, and a pharmaceutically acceptable carrier.

15. An immunoconjugate comprising the antibody, or antigen-binding portion thereof, of any one of claims 1-13 linked to a therapeutic agent.

16. The immunoconjugate of claim 15, wherein the therapeutic agent is a cytotoxin.

17. The immunoconjugate of claim 15, wherein the therapeutic agent is a radioisotope.

18. A composition comprising the immunoconjugate of any one of claims 15-17 and a pharmaceutically acceptable carrier.

19. An isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-13.

20. An expression vector comprising the nucleic acid molecule of claim 19.

21. A host cell comprising the expression vector of claim 20.

22. A method of making an anti-fucosyl-GM 1 antibody, comprising expressing the antibody in the host cell of claim 21, and isolating the antibody from the host cell.

23. Use of the antibody, or antigen-binding portion thereof, of any one of claims 1-13 in the manufacture of a medicament for treating a disease characterized by growth of fucosyl-GM 1-expressing tumor cells.

24. The use of claim 23, wherein the disease is cancer.

25. The use of claim 24, wherein the cancer is lung cancer.

26. The use of claim 25, wherein the lung cancer is small cell lung cancer.

27. The use of claim 25, wherein the lung cancer is non-small cell lung cancer.