Methods of upmodulating adaptive immune response using anti-PD-1 antibodies Number:7,521,051 from the United States Patent and Trademark Office (PTO) owispatent This disclosure provides antibodies and antigen-binding fragments that can act as agonists and/or antagonists of PD-1 (Programmed Death 1), thereby modulating immune responses in general, and those mediated by TcR and CD28, in particular. The disclosed compositions and methods may be used for example, in treating autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, and other immune system disorders.<H upmodulating, antibodies, Methods, of, upmo, 7521051.html, Patent, pto, 7521051

Title: Methods of upmodulating adaptive immune response using anti-PD-1 antibodies

Abstract: This disclosure provides antibodies and antigen-binding fragments that can act as agonists and/or antagonists of PD-1 (Programmed Death 1), thereby modulating immune responses in general, and those mediated by TcR and CD28, in particular. The disclosed compositions and methods may be used for example, in treating autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, and other immune system disorders.

Patent Number: 7,521,051 Issued on 04/21/2009 to Collins, et al.

Inventors: Collins; Mary (Natick, MA), Wood; Clive R. (Boston, MA), Carreno; Beatriz M. (Acton, MA), Luxenberg; Deborah (Melrose, MA), Jussif; Jason (Salem, NH), Carter; Laura L. (Medford, MA), Bennett; Frances K. (Sudbury, MA), Valge-Archer; Viia (Little Abington, GB), Andrews; John (Ware, GB), Russell; Caroline (Royston, GB)
Assignee: MedImmune Limited (Cambridge, GB)
Wyeth (Madison, NJ)

Appl. No.: 10/540,084

Filed: December 22, 2003

PCT Filed: December 22, 2003

PCT No.: PCT/IB03/06304

371(c)(1),(2),(4) Date: April 07, 2006

PCT Pub. No.: WO2004/056875

PCT Pub. Date: July 08, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60435354	Dec., 2002

Current U.S. Class: 424/144.1 ; 424/130.1; 424/141.1; 424/142.1; 424/143.1

Current International Class: A61K 39/395 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5837845	November 1998	Hirakawa et al.
6632927	October 2003	Adair et al.
6808710	October 2004	Wood et al.
7029674	April 2006	Carreno et al.
2004/0213795	October 2004	Collins et al.
2005/0180969	August 2005	Hardy et al.

Foreign Patent Documents


0 945 464	Sep., 1999	EP
WO 01/27272	Apr., 2001	WO
WO 02/078731	Oct., 2002	WO
WO 01/27279	Apr., 2004	WO
WO 2004/056875	Jul., 2004	WO

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Primary Examiner: Ouspenski; Ilia
Attorney, Agent or Firm: Wolf, Greenfield & Sack, P.C.

Parent Case Text

RELATED APPLICATIONS

This application is a national stage application of and claims priority under 35 USC .sctn. 371 to PCT Patent Application No. PCT/IB2003/006304, filed Dec. 22, 2003, which claims the benefit of U.S. Provisional Application No. 60/435,354 filed Dec. 23, 2002, both of which are incorporated herein by reference in their entirety.

Claims

We claim:

1. A method of upmodulating adaptive immune response comprising: contacting a lymphocyte with an isolated anti-PD-1 antibody, wherein the antibody comprises: a VH domain or an antigen-binding fragment thereof that comprises 3 CDRs; and a VL domain or an antigen-binding fragment thereof that comprises 3 CDRs; wherein the antibody comprises at least 3 VH domain CDRs having sequences of: SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25; or SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; or SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37; or at least 3 VL domain CDRs having sequences of: SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28; or SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34; or SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

2. The method of claim 1, wherein the antibody comprises a VH domain selected from the group consisting of SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:14; or a VL domain selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 12 and SEQ ID NO:16.

3. The method of claim 1, wherein the antibody specifically binds to the extracellular domain of PD-1 with an affinity constant greater than 10.sup.7 M.sup.-1.

4. The method of claim 1, wherein the antibody inhibits the binding of PD-L1 or PD-L2 to PD-1 with an IC.sub.50 of less than 10 nM.

5. The method of claim 1, wherein the antibody is a human antibody.

6. The method of claim 1, wherein the antibody is IgG.sub.1 or IgG.sub.4.

7. The method of claim 6, wherein the antibody is IgG.sub.1.lamda. or IgG.sub.1k.

8. The method of claim 1, wherein the antibody is selected from the group consisting of: an antibody comprising a VH domain of SEQ ID NO:6 and a VL domain of SEQ ID NO: 8; an antibody comprising a VH domain of SEQ ID NO:10 and a VL domain of SEQ ID NO:12; and an antibody comprising a VH domain of SEQ ID NO:14 and a VL domain of SEQ ID NO: 16.

9. The method of claim 1, wherein the lymphocyte is a T cell, B cell or monocyte.

10. The method of claim 1, wherein the antibody is immobilized on a support matrix or crosslinked.

11. The method of claim 1, wherein the support matrix comprises one or more material chosen from agarose, dextran, cellulose, PVDF, silica, nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, polyglycolic acid, polyhydroxyalkanoate, collagen and gelatin.

12. The method of claim 1, wherein the anti-PD-1 antibody modulates immune cell response mediated by an antigen receptor.

13. The method of claim 12, wherein the antigen receptor signal is co-presented with the anti-PD-1 antibody.

14. The method of claim 12, wherein the antigen receptor signal and anti-PD-1 antibody are spaced by no more than 100 .mu.m.

15. The method of claim 12, wherein the antigen receptor signal is delivered by an anti-CD3 antibody.

Description

DESCRIPTION OF THE INVENTION

1. Field of the Invention

The technical field relates to modulation of immune responses regulated by the Programmed Death 1 (PD-1) receptor.

2. Background of the Invention

An adaptive immune response involves activation, selection, and clonal proliferation of two major classes of lymphocytes termed T cells and B cells. After encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B cells proliferate and differentiate into antibody-secreting cells.

T cell activation is a multi-step process requiring several signaling events between the T cell and an antigen-presenting cell (APC). For T cell activation to occur, two types of signals must be delivered to a resting T cell. The first type is mediated by the antigen-specific T cell receptor (TcR), and confers specificity to the immune response. The second, costimulatory, type regulates the magnitude of the response and is delivered through accessory receptors on the T cell.

A primary costimulatory signal is delivered through the activating CD28 receptor upon engagement of its ligands B7-1 or B7-2. In contrast, engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2 ligands results in attenuation of T cell response. Thus, CTLA-4 signals antagonize costimulation mediated by CD28. At high antigen concentrations, CD28 costimulation overrides the CTLA-4 inhibitory effect. Temporal regulation of the CD28 and CTLA-4 expression maintains a balance between activating and inhibitory signals and ensures the development of an effective immune response, while safeguarding against the development of autoimmunity.

Molecular homologues of CD28 and CTLA-4 and their B-7 like ligands have been recently identified. ICOS is a CD28-like costimulatory receptor. PD-1 (Programmed Death 1) is an inhibitory receptor and a counterpart of CTLA-4. This disclosure relates to modulation of immune responses mediated by the PD-1 receptor.

PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).

PD-1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig V-like domain in its extracellular region. The PD-1 cytoplasmic domain contains two tyrosines, with the most membrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM (immuno-receptor tyrosine-based inhibitory motif). The presence of an ITIM on PD-1 indicates that this molecule functions to attenuate antigen receptor signaling by recruitment of cytoplasmic phosphatases. Human and murine PD-1 proteins share about 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues that define the Ig-V domain. The ITIM in the cytoplasmic region and the ITIM-like motif surrounding the carboxy-terminal tyrosine (TEYATI in human and mouse) are also conserved between human and murine orthologues.

PD-1 is expressed on activated T cells, B cells, and monocytes. Experimental data implicates the interactions of PD-1 with its ligands in downregulation of central and peripheral immune responses. In particular, proliferation in wild-type T cells but not in PD-1-deficient T cells is inhibited in the presence of PD-L1. Additionally, PD-1-deficient mice exhibit an autoimmune phenotype. PD-1 deficiency in the C57BL/6 mice results in chronic progressive lupus-like glomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart-tissue-specific self-reacting antibodies.

In general, a need exists to provide safe and effective therapeutic methods for immune disorders such as, for example, autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, immune deficiency, and other immune system-related disorders. Modulation of the immune responses involved in these disorders can be accomplished by manipulation of the PD-1 pathway.

SUMMARY OF THE INVENTION

The present disclosure provides antibodies that can act as agonists and/or antagonists of PD-1, thereby modulating immune responses regulated by PD-1. The disclosure further provides anti-PD-1 antibodies that comprise novel antigen-binding fragments. Anti-PD-1 antibodies of the invention are capable of (a) specifically binding to PD-1, including human PD-1; (b) blocking PD-1 interactions with its natural ligand(s); or (c) performing both functions. Furthermore, the antibodies may possess immunomodulatory properties, i.e., they may be effective in modulating the PD-1-associated downregulation of immune responses. Depending on the method of use and the desired effect, the antibodies may be used to either enhance or inhibit immune responses.

Nonlimiting illustrative embodiments of the antibodies are referred to as PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. Other embodiments comprise a V.sub.H and/or V.sub.L domain of the Fv fragment of PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2. Further embodiments comprise one or more complementarity determining regions (CDRs) of any of these V.sub.H and V.sub.L domains. Other embodiments comprise an H3 fragment of the V.sub.H domain of PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2.

The disclosure also provides compositions comprising PD-1 antibodies, and their use in methods of modulating immune response, including methods of treating humans or animals. In particular embodiments, anti-PD-1 antibodies are used to treat or prevent immune disorders by virtue of increasing or reducing the T cell response mediated by TcR/CD28. Disorders susceptible to treatment with compositions of the invention include but are not limited to rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosis, type I diabetes, transplant rejection, graft-versus-host disease, hyperproliferative immune disorders, cancer, and infectious diseases.

Additionally, anti-PD-1 antibodies may be used diagnostically to detect PD-1 or its fragments in a biological sample. The amount of PD-1 detected may be correlated with the expression level of PD-1, which, in turn, is correlated with the activation status of immune cells (e.g., activated T cells, B cells, and monocytes) in the subject.

The disclosure also provides isolated nucleic acids, which comprise a sequence encoding a V.sub.H or V.sub.L domain from the Fv fragment of PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2. Also provided are isolated nucleic acids, which comprise a sequence encoding one or more CDRs from any of the presently disclosed V.sub.H and V.sub.L domains. The disclosure also provides vectors and host cells comprising such nucleic acids.

The disclosure further provides a method of producing new V.sub.H and V.sub.L domains and/or functional antibodies comprising all or a portion of such domains derived from the V.sub.H or V.sub.L domains of PD1-17, PD1-28, PD1-33, PD1-35, or PD1-F2.

Additional aspects of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practicing the invention. The invention is set forth and particularly pointed out in the appended claims, and the present disclosure should not be construed as limiting the scope of the claims in any way. The following detailed description includes exemplary representations of various embodiments of the invention, which are not restrictive of the invention, as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate various embodiments and not limit the invention. Citation of references is not an admission that these references are prior art to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show reactivity of scFv antibodies with human PD-1 as determined by phage ELISA.

FIGS. 2A-2C show reactivity of IgG-converted antibodies with human or mouse PD-1 as determined by ELISA.

FIG. 3 shows results of an ELISA demonstrating that selected PD-1 antibodies inhibit binding of PD-L1 to PD-1.

FIG. 4 shows results of an ELISA demonstrating that immunomodulatory PD-1 antibodies bind to distinct sites on PD-1 as determined by cross-blocking ELISA assays.

FIG. 5 shows results of T-cell proliferation assays demonstrating that co-engagement by TcR and anti-PD-1 antibody PD1-17 or PD-L1.Fc reduces proliferation. Co-engagement by TcR and anti-PD-1 J110 has no effect on proliferation.

FIG. 6 demonstrates enhanced proliferation of primary T cells by PD1-17 in a soluble form.

DETAILED DESCRIPTION

Definitions

The term "antibody," as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact," as in "intact antibodies," for the purposes of this disclosure, the term "antibody" also includes antibody fragments such as Fab, F(ab').sub.2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind PD-1 specifically. Typically, such fragments would comprise an antigen-binding domain.

The terms "antigen-binding domain," "antigen-binding fragment," and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant."

An antigen-binding domain typically comprises an antibody light chain variable region (V.sub.L) and an antibody heavy chain variable region (V.sub.H), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a V.sub.H domain, but still retains some antigen-binding function of the intact antibody.

The term "repertoire" refers to a genetically diverse collection of nucleotides derived wholly or partially from sequences that encode expressed immunoglobulins. The sequences are generated by in vivo rearrangement of, e.g., V, D, and J segments for H chains and, e.g., V and J segment for L chains. Alternatively, the sequences may be generated from a cell line by in vitro stimulation, in response to which the rearrangement occurs. Alternatively, part or all of the sequences may be obtained by combining, e.g., unrearranged V segments with D and J segments, by nucleotide synthesis, randomised mutagenesis, and other methods, e.g., as disclosed in U.S. Pat. No. 5,565,332.

The terms "specific interaction" and "specific binding" refer to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant K.sub.A is higher than 10.sup.6 M.sup.-1, or more preferably higher than 10.sup.8 M.sup.-1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques. Illustrative conditions are set forth in Examples 1, 2, 4, 6, and 7.

The phrase "substantially as set out" means that the relevant CDR, V.sub.H, or V.sub.L domain of the invention will be either identical to or have only insubstantial differences in the specified regions (e.g., a CDR), the sequence of which is set out. Insubstantial differences include minor amino acid changes, such as substitutions of 1 or 2 out of any 5 amino acids in the sequence of a specified region.

The term "PD-1 activity" refers to one or more immunoregulatory activities associated with PD-1. For example, PD-1 is a negative regulator of the TcR/CD28-mediated immune response. Procedures for assessing the PD-1 activity in vivo and in vitro are described in Examples 8, 9, and 10.

The terms "modulate," "immunomodulatory," and their cognates refer to a reduction or an increase in the activity of PD-1 associated with downregulation of T cell responses due to its interaction with an anti-PD-1 antibody, wherein the reduction or increase is relative to the activity of PD-1 in the absence of the same antibody. A reduction or an increase in activity is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. When PD-1 activity is reduced, the terms "modulatory" and "modulate" are interchangeable with the terms "inhibitory" and "inhibit." When PD-1 activity is increased, the terms "modulatory" and "modulate" are interchangeable with the terms "activating" and "activate." The activity of PD-1 can be determined quantitatively using T cell proliferation assays as described in Examples 8 and 9.

The terms "treatment" and "therapeutic method" refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures).

The term "effective amount" refers to a dosage or amount that is sufficient to reduce the activity of PD-1 to result in amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., increased cytolytic activity of T cells, induction of immune tolerance, reduction or increase of the PD-1 activity associated with the negative regulation of T-cell mediated immune response, etc.

The term "isolated" refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to preparations where the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or at least 70-80% (w/w) pure, more preferably, at least 80-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

Anti-PD-1 Antibodies

The disclosure provides anti-PD-1 antibodies that comprise novel antigen-binding fragments.

In general, antibodies can be made, for example, using traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display performed with antibody libraries (Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597). For other antibody production techniques, see also Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The invention is not limited to any particular source, species of origin, method of production.

Intact antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, designated as the .lamda. chain and the .kappa. chain, are found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2.

The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Harlow et al., supra. Briefly, each light chain is composed of an N-terminal variable domain (V.sub.L) and a constant domain (C.sub.L). Each heavy chain is composed of an N-terminal variable domain (V.sub.H), three or four constant domains (C.sub.H), and a hinge region. The C.sub.H domain most proximal to V.sub.H is designated as C.sub.H1. The V.sub.H and V.sub.L domains consist of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDRs). The CDRs contain most of the residues responsible for specific interactions with the antigen. The three CDRs are referred to as CDR1, CDR2, and CDR3. CDR constituents on the heavy chain are referred to as H1, H2, and H3, while CDR constituents on the light chain are referred to as L1, L2, and L3, accordingly. CDR3 and, particularly H3, are the greatest source of molecular diversity within the antigen-binding domain. H3, for example, can be as short as two amino acid residues or greater than 26.

The Fab fragment (Fragment antigen-binding) consists of the V.sub.H-C.sub.H1 and V.sub.L-C.sub.L domains covalently linked by a disulfide bond between the constant regions. To overcome the tendency of non-covalently linked V.sub.H and V.sub.L domains in the Fv to dissociate when co-expressed in a host cell, a so-called single chain (sc) Fv fragment (scFv) can be constructed. In a scFv, a flexible and adequately long polypeptide links either the C-terminus of the V.sub.H to the N-terminus of the V.sub.L or the C-terminus of the V.sub.L to the N-terminus of the V.sub.H. Most commonly, a 15-residue (Gly.sub.4Ser).sub.3 peptide is used as a linker but other linkers are also known in the art.

Antibody diversity is a result of combinatorial assembly of multiple germline genes encoding variable regions and a variety of somatic events. The somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete V.sub.H region and the recombination of variable and joining gene segments to make a complete V.sub.L region. The recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions. These mechanisms of diversity occur in the developing B cell prior to antigen exposure. After antigenic stimulation, the expressed antibody genes in B cells undergo somatic mutation.

Based on the estimated number of germline gene segments, the random recombination of these segments, and random V.sub.H-V.sub.L pairing, up to 1.6.times.10.sup.7 different antibodies could be produced (Fundamental Immunology, 3.sup.rd ed., ed. Paul, Raven Press, New York, N.Y., 1993). When other processes which contribute to antibody diversity (such as somatic mutation) are taken into account, it is thought that upwards of 1.times.10.sup.10 different antibodies could be potentially generated (Immunoglobulin Genes, 2.sup.nd ed., eds. Jonio et al., Academic Press, San Diego, Calif., 1995). Because of the many processes involved in antibody diversity, it is highly unlikely that independently generated antibodies will have identical or even substantially similar amino acid sequences in the CDRs.

The disclosure provides novel CDRs derived from human immunoglobulin gene libraries. The structure for carrying a CDR will generally be an antibody heavy or light chain or a portion thereof, in which the CDR is located at a location corresponding to the CDR of naturally occurring V.sub.H and V.sub.L. The structures and locations of immunoglobulin variable domains may be determined, for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.

DNA and amino acid sequences of anti-PD-1 antibodies, their scFv fragment, V.sub.H and V.sub.L domains, and CDRs are set forth in the Sequence Listing and are enumerated as listed in Table 1. Particular nonlimiting illustrative embodiments of the antibodies are referred to as PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. The positions for each CDR within the V.sub.H and V.sub.L domains of the illustrative embodiments are listed in Tables 2 and 3.

TABLE-US-00001 TABLE 1 DNA and Amino Acid (AA) Sequences of V.sub.H and V.sub.L Domains and CDRs Sequence PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2 V.sub.H DNA SEQ ID NO: 1 SEQ ID NO: 5 SEQ ID NO: 9 SEQ ID NO: 13 SEQ ID NO: 46 V.sub.H AA SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 10 SEQ ID NO: 14 SEQ ID NO: 47 V.sub.L DNA SEQ ID NO: 3 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 15 SEQ ID NO: 48 V.sub.L AA SEQ ID NO: 4 SEQ ID NO: 8 SEQ ID NO: 12 SEQ ID NO: 16 SEQ ID NO: 49 H1 AA SEQ ID NO: 17 SEQ ID NO: 23 SEQ ID NO: 29 SEQ ID NO: 35 SEQ ID NO: 50 H2 AA SEQ ID NO: 18 SEQ ID NO: 24 SEQ ID NO: 30 SEQ ID NO: 36 SEQ ID NO: 51 H3 AA SEQ ID NO: 19 SEQ ID NO: 25 SEQ ID NO: 31 SEQ ID NO: 37 SEQ ID NO: 52 L1 AA SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 32 SEQ ID NO: 38 SEQ ID NO: 53 L2 AA SEQ ID NO: 21 SEQ ID NO: 27 SEQ ID NO: 33 SEQ ID NO: 39 SEQ ID NO: 54 L3 AA SEQ ID NO: 22 SEQ ID NO: 28 SEQ ID NO: 34 SEQ ID NO: 40 SEQ ID NO: 55

TABLE-US-00002 TABLE 2 Positions of Heavy Chain CDRs PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID CDR NO: 2 NO: 6 NO: 10 NO: 14 NO: 47 H1 31-42 31-35 31-35 31-37 34-42 H2 57-72 50-66 50-66 52-67 57-73 H3 105-117 99-108 99-108 100-116 106-114

TABLE-US-00003 TABLE 3 Positions of Light Chain CDRs PD1-17 PD1-28 PD1-33 PD1-35 PD1-F2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID CDR NO: 4 NO: 8 NO: 12 NO: 16 NO: 49 L1 23-35 23-33 23-36 23-35 28-35 L2 51-57 49-55 52-58 51-57 54-61 L3 92-100 88-98 91-102 90-100 94-101

Anti-PD-1 antibodies may optionally comprise antibody constant regions or parts thereof. For example, a V.sub.L domain may have attached, at its C terminus, antibody light chain constant domains including human C.kappa. or C.lamda. chains. Similarly, a specific antigen-binding domain based on a V.sub.H domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g., IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgG.sub.1 and IgG.sub.4. In the exemplary embodiments, PD1-17, PD1-28, PD1-33, and PD1-35, antibodies comprise C-terminal fragments of heavy and light chains of human IgG.sub.1.lamda., while PD1-F2 comprises C-terminal fragments of heavy and light chains of human IgG.sub.1.kappa.. The DNA and amino acid sequences for the C-terminal fragment of are well known in the art (see, e.g, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991). Nonlimiting exemplary sequences are set forth in Table 4.

TABLE-US-00004 TABLE 4 C-Terminal Region DNA Amino acid IgG1 heavy chain SEQ ID NO: 44 SEQ ID NO: 45 .lamda. light chain SEQ ID NO: 42 SEQ ID NO: 43 .kappa. light chain SEQ ID NO: 57 SEQ ID NO: 58

Certain embodiments comprise a V.sub.H and/or V.sub.L domain of an Fv fragment from PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2. Further embodiments comprise at least one CDR of any of these V.sub.H and V.sub.L domains. Antibodies, comprising at least one of the CDR sequences set out in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:16-40, SEQ ID NO:47, or SEQ ID NO:49 are encompassed within the scope of this invention. An embodiment, for example, comprises an H3 fragment of the V.sub.H domain of antibodies chosen from at least one of PD1-17, PD1-28, PD1-33, PD1-35, and PD1-F2.

In certain embodiments, the V.sub.H and/or V.sub.L domains may be germlined, i.e., the framework regions (FRs) of these domains are mutated using conventional molecular biology techniques to match those produced by the germline cells. In other embodiments, the framework sequences remain diverged from the consensus germline sequences.

In certain embodiments, the antibodies specifically bind an epitope within the extracellular domain of human PD-1. The predicted extracellular domain consists of a sequence from about amino acid 21 to about amino acid 170 of SEQ ID NO:41 (Swissport Accession No. Q15116). In certain other embodiments, the antibodies specifically bind an epitope within the extracellular domain of mouse PD-1, with an affinity of more than 10.sup.7 M.sup.-1, and preferably more than 10.sup.8 M.sup.-1. The amino acid sequence of mouse PD-1 is set out in SEQ ID NO:56 (Accession No. NM.sub.--008798) and is as a whole about 60% identical to its human counterpart. In further embodiments, antibodies of the invention bind to the PD-L-binding domain of PD-1.

It is contemplated that antibodies of the invention may also bind with other proteins, including, for example, recombinant proteins comprising all or a portion of the PD-1 extracellular domain.

One of ordinary skill in the art will recognize that the antibodies of this invention may be used to detect, measure, and inhibit proteins that differ somewhat from PD-1. The antibodies are expected to retain the specificity of binding so long as the target protein comprises a sequence which is at least about 60%, 70%, 80%, 90%, 95%, or more identical to any sequence of at least 100, 80, 60, 40, or 20 of contiguous amino acids in the sequence set forth SEQ ID NO:41. The percent identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48: 444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4: 11-17.

In addition to the sequence homology analyses, epitope mapping (see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996) and secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Derivatives

This disclosure also provides a method for obtaining an antibody specific for PD-1. CDRs in such antibodies are not limited to the specific sequences of V.sub.H and V.sub.L identified in Table 1 and may include variants of these sequences that retain the ability to specifically bind PD-1. Such variants may be derived from the sequences listed in Table 1 by a skilled artisan using techniques well known in the art. For example, amino acid substitutions, deletions, or additions, can be made in the FRs and/or in the CDRs. While changes in the FRs are usually designed to improve stability and immunogenicity of the antibody, changes in the CDRs are typically designed to increase affinity of the antibody for its target. Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes may be determined empirically by routine techniques that involve altering the CDR and testing the affinity antibody for its target. For example, conservative amino acid substitutions can made within any one of the disclosed CDRs. Various alterations can be made according to the methods described in Antibody Engineering, 2.sup.nd ed., Oxford University Press, ed. Borrebaeck, 1995. These include but are not limited to nucleotide sequences that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a "silent" change. For example, the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 5). Furthermore, any native residue in the polypeptide may also be substituted with alanine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al. (1998) Adv. Biophys. 35:1-24).

Derivatives and analogs of antibodies of the invention can be produced by various techniques well known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2.sup.nd ed., Spring Verlag, Berlin, Germany).

TABLE-US-00005 TABLE 5 Original Exemplary Typical Residues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K) Arg, 1,4-Diamino-butyric Arg Acid, Gln, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, Leu Norleucine

In one embodiment, a method for making a V.sub.H domain which is an amino acid sequence variant of a V.sub.H domain of the invention comprises a step of adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of the presently disclosed V.sub.H domain, optionally combining the V.sub.H domain thus provided with one or more V.sub.L domains, and testing the V.sub.H domain or V.sub.H/V.sub.L combination or combinations for a specific binding to PD-1 or and, optionally, testing the ability of such antigen-binding domain to modulate PD-1 activity. The V.sub.L domain may have an amino acid sequence that is identical or is substantially as set out according to Table 1.

An analogous method can be employed in which one or more sequence variants of a V.sub.L domain disclosed herein are combined with one or more V.sub.H domains.

A further aspect of the disclosure provides a method of preparing antigen-binding fragment that specifically binds with PD-1. The method comprises: (a) providing a starting repertoire of nucleic acids encoding a V.sub.H domain that either includes a CDR3 to be replaced or lacks a CDR3 encoding region; (b) combining the repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for a V.sub.H CDR3 (i.e., H3) such that the donor nucleic acid is inserted into the CDR3 region in the repertoire, so as to provide a product repertoire of nucleic acids encoding a V.sub.H domain; (c) expressing the nucleic acids of the product repertoire; (d) selecting a binding fragment specific for PD-1; and (e) recovering the specific binding fragment or nucleic acid encoding it.

Again, an analogous method may be employed in which a V.sub.L CDR3 (i.e., L3) of the invention is combined with a repertoire of nucleic acids encoding a V.sub.L domain, which either include a CDR3 to be replaced or lack a CDR3 encoding region. The donor nucleic acid may be selected from nucleic acids encoding an amino acid sequence substantially as set out in SEQ ID NO:17-40 or SEQ ID NO:50-55.

A sequence encoding a CDR of the invention (e.g., CDR3) may be introduced into a repertoire of variable domains lacking the respective CDR (e.g., CDR3), using recombinant DNA technology, for example, using methodology described by Marks et al. (Bio/Technology (1992) 10: 779-783). In particular, consensus primers directed at or adjacent to the 5' end of the variable domain area can be used in conjunction with consensus primers to the third framework region of human V.sub.H genes to provide a repertoire of V.sub.H variable domains lacking a CDR3. The repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences may be shuffled with repertoires of V.sub.H or V.sub.L domains lacking a CDR3, and the shuffled complete V.sub.H or V.sub.L domains combined with a cognate V.sub.L or V.sub.H domain to make the PD-1-specific antibodies of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system such as described in WO92/01047 so that suitable antigen-binding fragments can be selected.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature (1994) 370: 389-391), who describes the technique in relation to a .beta.-lactamase gene but observes that the approach may be used for the generation of antibodies.

In further embodiments, one may generate novel V.sub.H or V.sub.L regions carrying one or more sequences derived from the sequences disclosed herein using random mutagenesis of one or more selected V.sub.H and/or V.sub.L genes. One such technique, error-prone PCR, is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).

Another method that may be used is to direct mutagenesis to CDRs of V.sub.H or V.sub.L genes. Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263: 551-567).

Similarly, one or more, or all three CDRs may be grafted into a repertoire of V.sub.H or V.sub.L domains, which are then screened for an antigen-binding fragment specific for PD-1.

A portion of an immunoglobulin variable domain will comprise at least one of the CDRs substantially as set out herein and, optionally, intervening framework regions from the scF.sub.v fragments as set out herein. The portion may include at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains to further protein sequences including immunoglobulin heavy chain constant regions, other variable domains (for example, in the production of diabodies), or proteinaceous labels as discussed in further detail below.

Although the embodiments illustrated in the Examples comprise a "matching" pair of V.sub.H and V.sub.L domains, a skilled artisan will recognize that alternative embodiments may comprise antigen-binding fragments containing only a single CDR from either V.sub.L or V.sub.H domain. Either one of the single chain specific binding domains can be used to screen for complementary domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to PD-1. The screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO92/01047, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding domain is selected in accordance with phage display techniques as described.

Anti-PD1 antibodies described herein can be linked to another functional molecule, e.g., another peptide or protein (albumin, another antibody, etc.), toxin, radioisotope, cytotoxic or cytostatic agents. For example, the antibodies can be linked by chemical cross-linking or by recombinant methods. The antibodies may also be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods to attach them are also shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

The disclosed antibodies may also be altered to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties can be deleted and/or one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. Such methods are described in WO 87/05330 and in Aplin et al. (1981) CRC Crit. Rev. Biochem., 22: 259-306. Removal of any carbohydrate moieties from the antibodies may be accomplished chemically or enzymatically, for example, as described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259: 52; and Edge et al. (1981) Anal. Biochem., 118:131 and by Thotakura et al. (1987) Meth. Enzymol., 138: 350. The antibodies may also be tagged with a detectable, or functional, label. Detectable labels include radiolabels such as .sup.131I or .sup.99Tc, which may also be attached to antibodies using conventional chemistry. Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

Antibodies, in which CDR sequences differ only insubstantially from those set out in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NOs:16-40, SEQ ID NO:47, or SEQ ID NO:49 are encompassed within the scope of this invention. Typically, an amino acid is substituted by a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions would be within the ordinary skills of an artisan. Unlike in CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to, humanizing a non-human derived or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter the effector function such as Fc receptor binding, e.g., as described in U.S. Pat. Nos. 5,624,821 and 5,648,260 and Lund et al. (1991) J. Immun. 147: 2657-2662 and Morgan et al. (1995) Immunology 86: 319-324, or changing the species from which the constant region is derived.

One of skill in the art will appreciate that the modifications described above are not all-exhaustive, and that many other modifications would obvious to a skilled artisan in light of the teachings of the present disclosure.

Nucleic Acids, Cloning and Expression Systems

The present disclosure further provides isolated nucleic acids encoding the disclosed antibodies. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

The nucleic acids provided herein comprise a coding sequence for a CDR, a V.sub.H domain, and/or a V.sub.L domain disclosed herein.

The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes which comprise at least one nucleic acid encoding a CDR, a V.sub.H domain, and/or a V.sub.L domain disclosed here.

The disclosure further provides a host cell which comprises one or more constructs as above.

Also provided are nucleic acids encoding any CDR (H1, H2, H3, L1, L2, or L3), V.sub.H or V.sub.L domain, as well as methods of making of the encoded products. The method comprises expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a V.sub.H or V.sub.L domain, or specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

Antigen-binding fragments, V.sub.H and/or V.sub.L domains, and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the invention may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, 2.sup.nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

A further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed here. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.

Methods of Use

The disclosed anti-PD-1 antibodies are capable of modulating the PD-1-associated downregulation of the immune responses. In particular embodiments, the immune response is TcR/CD28-mediated. The disclosed antibodies can act as either agonists or antagonists of PD-1, depending on the method of their use. The antibodies can be used to prevent, diagnose, or treat medical disorders in mammals, especially, in humans. Antibodies of the invention can also be used for isolating PD-1 or PD-1-expressing cells. Furthermore, the antibodies can be used to treat a subject at risk of or susceptible to a disorder or having a disorder associated with aberrant PD-1 expression or function.

Antibodies of the invention can be used in methods for induction of tolerance to a specific antigen (e.g., a therapeutic protein). In one embodiment, tolerance is induced against a specific antigen by co-administration of antigen and an anti-PD-1 antibody of the invention. For example, patients that received Factor VIII frequently generate antibodies to this protein; co-administration of an anti-PD-1 antibody of the invention in combination with recombinant Factor VIII is expected to result in the downregulation of immune responses to this clotting factor.

Antibodies of the invention can be used in circumstances where a reduction in the level of immune response may be desirable, for example, in certain types of allergy or allergic reactions (e.g., by inhibition of IgE production), autoimmune diseases (e.g., rheumatoid arthritis, type I diabetes mellitus, multiple sclerosis, inflammatory bowel disease, Crohn's disease, and systemic lupus erythematosis), tissue, skin and organ transplant rej