阅读说明：本技术 用于免疫疗法的组合物和方法 (Compositions and methods for immunotherapy ) 是由 布鲁斯·麦克里迪 蒂莫西·戴维·琼斯 弗朗西斯·约瑟夫·卡尔 于 2014-06-23 设计创作，主要内容包括：本发明提供用于免疫疗法的组合物和方法,其包括用于诱导抗原特异性T细胞的货架稳定的药物组合物。此类组合物用作人工抗原呈递细胞(aAPC)的组分,以向患者提供复合物用于呈递抗原(如,肿瘤抗原)和/或T细胞共刺激分子。(The present invention provides compositions and methods for immunotherapy comprising a shelf-stable pharmaceutical composition for inducing antigen-specific T cells. Such compositions are useful as components of artificial antigen presenting cells (aapcs) to provide complexes to patients for presentation of antigens (e.g., tumor antigens) and/or T cell co-stimulatory molecules.)

1. A pharmaceutical composition comprising polymeric particles having an anti-CD 28 antibody and an antigen presenting complex ligand attached thereto, wherein,

the anti-CD 28 antibody is a co-stimulatory ligand having an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region,

the immunoglobulin heavy chain variable region amino acid sequence is as follows: EVKLQQSGPGLVKPSETLSLTCTVSGFSLSDYGVHWVRQAPGKGLEWLGVIWAGGGTNYNSALMSRKTISKDNSKSQVSLKMSSVTAADTAVYYCARDKGYSYYYSMDYWGQGTLVTVSS the flow of the air in the air conditioner,

the variable region amino acid sequence of the immunoglobulin light chain is as follows: DIELTQSPDSLAVSLGERATINCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAMYFCQQSRKVPYTFGGGTKVEIK are provided.

2. The pharmaceutical composition of claim 1, wherein the antigen presenting complex comprises an MHC class I and/or MHC class II complex.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is co-formulated with an antigenic peptide for presentation to a T cell.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the MHC class I and/or II complex comprises a peptide bound to an antigen binding pocket.

5. The pharmaceutical composition of claim 4, wherein the peptide is a tumor associated antigen.

6. The pharmaceutical composition of any one of claims 1-5, wherein the antigen presenting complex comprises an HLA amino acid sequence.

7. The pharmaceutical composition of claim 6, wherein the HLA amino acid sequence is HLA-A02: 01.

8. The pharmaceutical composition of any one of claims 1 to 6, wherein the anti-CD 28 antibody is not a superagonist.

9. The pharmaceutical composition of any one of claims 1-8, wherein the immunoglobulin heavy chain amino acid sequence comprises an IgG4 constant region sequence.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the antigen presenting complex and co-stimulatory ligand are present in a ratio of 5:1 to 1: the ratio of 5 is conjugated.

11. The pharmaceutical composition of any one of claims 1-10, wherein the pharmaceutical composition is formulated for intravenous, intraarterial, subcutaneous, intradermal, intralymphatic, or intratumoral administration.

Technical Field

The present invention relates to compositions (including pharmaceutical compositions) and methods for immunotherapy.

Background

An Antigen Presenting Cell (APC) is a cell that processes and presents antigenic peptides complexed with Major Histocompatibility Complex (MHC) proteins on its surface. Effector cells, such as T cells, can recognize these peptide-mhc (pmhc) complexes using receptors such as T Cell Receptors (TCRs).

Dendritic Cells (DCs) are an example of an antigen presenting cell that can be stimulated to efficiently present antigen and support immune effector cell expansion, thereby activating a cytotoxic response to the antigen. In some immunotherapies, DCs are collected from a patient and pulsed with an antigen or transfected with a viral vector. Upon return to the patient, these activated cells present tumor antigens to effector lymphocytes (e.g., CD 4)⁺T cell, CD8⁺T cells and B cells). If appropriate, the therapy can initiate a cytotoxic response against cells expressing the antigen, including tumor antigens.

However, DC immunotherapy, such as many immunotherapies, face significant limitations. For example, ex vivo detectable strong and antigen-specific T cell responses in vaccinated cancer patients differ from only weak clinical responses. Janikashvili N et al, Personalized dentritic cell-based tumor immunotherapy, immunotherapy 2010, 1 month 1; 2(1):57.

There remains a need for compositions (including shelf-stable pharmaceutical compositions) and methods effective for immunotherapy, including antigen-specific immunotherapy.

Summary of The Invention

The present invention provides compositions and methods for immunotherapy comprising a shelf-stable pharmaceutical composition for inducing antigen-specific T cells in a patient. Such compositions are useful for treating, for example, cancer and infectious diseases. In some aspects, the compositions are artificial antigen presenting cells (aapcs) comprising pharmaceutically acceptable beads or particles having on their surface an antigen presenting complex and optionally a T cell costimulatory signal to provide a patient with a molecular complex that presents one or more antigens (e.g., one or more tumor antigens) under appropriate circumstances to activate antigen-specific T cells. The beads or particles are designed to provide pharmacodynamic advantages, including cycling characteristics, biodistribution and degradation kinetics, and activity. Such parameters include size, surface charge, polymer composition, ligand conjugation chemistry, ligand density, and the like.

In some embodiments, the T cell co-stimulatory signal is an anti-CD 28 antibody or portion thereof, which may comprise human heavy chain amino acid sequences, including sequences selected from IgG, IgD, IgA, or IgM isotypes. In some embodiments, the immunoglobulin sequences comprise human IgG constant and variable sequences. Framework (FW) sequences can be modified to contain important or desirable murine framework residues to maintain the integrity of one or more antigen binding sites. Complementarity Determining Regions (CDRs) can be based on murine antibody amino acid sequences (e.g., 9.3mAb), or other CD28 binding sequences, many of which are known. In some embodiments, the antibody heavy chain is a variant of human IGHV4 (e.g., IGHV4-59) germline FW. In some embodiments, the antibody comprises a light chain and the light chain is a variant of human IGKV4-01 FW. The antibody may comprise a constant region, and the constant region may be human IgG4 or a variant thereof.

The co-stimulatory molecule can be conjugated to a solid support with a complex of antigen presenting molecules to induce antigen-specific T cells. The antigen presenting molecule complex may comprise MHC class I and/or class II complexes, or portions thereof comprising antigen binding grooves. In some embodiments, the molecular complex comprises one or more HLA amino acid sequences (e.g., comprising the extracellular domain of HLA or antigen presenting portion thereof), which may comprise additional sequences, such as immunoglobulin sequences, or other dimerization or stabilization sequences. In some embodiments, HLA-Ig dimerization fusions provide advantages in stability and/or binding affinity.

Thus, in some embodiments, the present invention provides bead-or particle-conjugated molecular complexes for presenting antigens to T cells, wherein the complexes comprise an amino acid sequence, or portion thereof, that forms a class I or class II antigen binding pocket. The amino acid sequence of the antigen presenting complex may include a fusion with a heterologous sequence to provide, for example, stability, affinity, and steric advantages. In some embodiments, the heterologous sequence comprises an immunoglobulin sequence. In some embodiments, the molecular complex comprises an HLA (e.g., HLA-a2) amino acid sequence fused to a heterologous sequence, such as an immunoglobulin sequence. In some embodiments, the immunoglobulin comprises a human heavy chain immunoglobulin sequence (e.g., IGVH4), which may include an immunoglobulin constant sequence to provide a dimeric HLA, and may optionally comprise a variable region sequence. The variable sequences, if present, can be optionally modified to reduce or eliminate potential antigen binding and optionally are free of murine FW residues. The HLA amino acid sequence may be HLA-a 02:01(IMGT accession number HLA00005) or a derivative thereof.

T cell co-stimulatory ligands and/or antigen presenting complexes (as well as other ligands described herein, including targeting ligands) may be conjugated to solid supports for antigen presentation and antigen-specific T cell activation ex vivo or in vivo. In some embodiments, the solid support is a bead or particle (e.g., a PLGA or PLGA-PEG particle) having surface functional groups for coupling ligands. The particles are designed to provide pharmacodynamic advantages, including cycling characteristics, biodistribution and degradation kinetics, and activity. Such parameters include size, surface charge, polymer composition, ligand conjugation chemistry, ligand density, and the like.

In various embodiments, the pharmaceutical composition may further comprise an antigenic peptide for presentation to T cells and may be co-formulated with the ligand-conjugated beads or particles. In various embodiments, the pharmaceutical composition is shelf-stable and may be provided in a lyophilized form for reconstitution prior to administration, or alternatively in another convenient form for administration to a patient (e.g., by parenteral administration).

The pharmaceutical compositions described herein are used in immunotherapy, e.g., methods for inducing antigen-specific cytotoxic T cell formation, by administering an effective amount of the composition to a patient in need thereof. In particular, the antigen presentation platform may be used to treat patients with infectious diseases, cancer or autoimmune diseases, or to provide prophylactic protection to immunosuppressed patients.

The invention also provides polynucleotides encoding the amino acid sequences described herein, as well as host cells expressing the amino acid sequences described herein.

The invention is further illustrated by the following non-limiting examples.

The details of the invention are set forth in the following description and claims. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Brief Description of Drawings

Three humanized variable heavy sequences of anti-CD 28 are shown in fig. 1-3.

FIGS. 4-6 show three humanized variable light sequences of anti-CD 28.

Figure 7 shows the modified constant heavy sequence.

FIG. 8 shows constant kappa light sequences.

Figure 9 shows the humanized non-CD 28-binding variable regions used to construct HLA fusions.

FIG. 10 shows the amino acid sequence of humanized HLA-IgG4 HC.

FIG. 11 shows the amino acid sequence of light chain 3(LC3 or V κ 3).

Figure 12 shows the amino acid sequence of heavy chain 1(HC 1).

Figure 13 shows the amino acid sequence of heavy chain 2(HC 2).

FIGS. 14-16 show expression constructs for expression in the STABLENFAST-NS 0 cell line.

Figure 17 shows that the humanized anti-CD 28 mAb is not a superagonist.

FIG. 18 shows humanized anti-CD 28 clone specifically stains CD28 on human T-cell line. Fig. 18 (a): staining with murine anti-human CD8 mAb (clone 9.3, isotype IgG2 a); fig. 18 (B): staining was with humanized anti-CD 28 (isotype IgG 4).

Detailed Description

The following abbreviations may be used throughout: BLAST-basic local alignment search tool, CDR-complementarity determining regions, ck-kappa light chain constant regions, Fc-antibody fragment crystallizable regions, Fw- (of the variable region) framework regions, HLA-human leukocyte antigens, MHC-major histocompatibility complex, VH-variable heavy chains, vk-variable kappa light chains and V region-antibody variable regions, VH or vk.

The present invention provides compositions and methods for immunotherapy comprising a shelf-stable pharmaceutical composition for inducing antigen-specific T cells in a patient. In some embodiments, the composition comprises a dimeric HLA antigen presenting complex. In some embodiments, the compositions comprise humanized immunoglobulin sequences or portions thereof that can be used as components of ligands on artificial antigen presenting cells (aapcs) to provide dimeric molecular complexes to a patient for presentation of one or more antigens (e.g., one or more tumor antigens) and optionally one or more co-stimulatory signals. The antigen presentation platform, as described in more detail below, may be based on an artificial solid support, such as a pharmaceutically acceptable support (including latex or polymer beads or particles).

In some embodiments, the T cell co-stimulatory signal is an anti-CD 28 antibody or portion thereof. In some embodiments, the anti-CD 28 antibody comprises a sequence of at least one human immunoglobulin isotype selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA, or IgM. For example, the anti-CD 28 antibody can be of the IgG isotype and can contain the sequence of one or more IgG germline framework sequences. For example, the anti-CD 28 may contain the human IGHV4 heavy chain amino acid sequence, which may be modified to have one to fifteen amino acid modifications. The modifications can include murine framework residues to support the integrity of one or more antigen binding sites.

In some embodiments, the Complementarity Determining Regions (CDRs) are based on murine antibody amino acid sequences, which may optionally comprise one to ten, such as one to five, amino acid modifications. In some embodiments, one, two, three or more CDRs are based on the mouse 9.3mAb (Tan et al j. exp. med.1993177:165), which is publicly available. Exemplary CDRs are shown in fig. 1-6. In some embodiments, the antibody has a full set of heavy chain and/or a full set of light chain CDRs (9.3 mAb). For example, in some embodiments, the heavy chain variable region comprises one, two, or three of the following CDRs, each of which may be optionally modified with one, two, or three amino acid substitutions, deletions, or additions: CDR1(DYGVH), CDR2(VIWAGGGTNYNSALMS), and CDR3 (DKGYSYYYSMDY). In some embodiments, the light chain variable region comprises one, two, or three of the following CDRs, each of which may be modified by one, two, or three amino acid substitutions, deletions, or additions: CDR1(RASESVEYYVTSLMQ), CDR2(AASNVES), and CDR3 (QQSRKVPYT).

Alternative CDR sequences, variable regions or CD 28-binding ligands may be used in various embodiments. Alternative ligands and antibodies are described in U.S. patent 7,612,170, U.S. patent 6,987,171, and U.S. patent 6,887,466, for example, and these disclosures are hereby incorporated by reference in their entirety.

In some embodiments, the antibody heavy chain comprises a variant of a human IGHV4-59 germline Framework (FW) modified to include 5 to 15 murine FW residues. In some embodiments, the antibody comprises a light chain amino acid sequence, and the light chain sequence can be a variant of a human IGKV4-01FW sequence, and which can be modified to include 3 to 15 murine FW residues.

The anti-CD 28 human heavy chain sequence may be modified, for example, to include one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all) murine Fw residues at positions 1, 3, 6, 37, 48, 67, 71, 73, 76, 78, 82a, and 82c (based on Kabat numbering). The murine Fw residues at these positions can be as in 9.3 mAb. The light chain can be modified to include one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or all) murine Fw residues at positions 3, 4, 49, 70, 85, 87, and 80. Selected murine Fw residues may support the integrity of the antigen-binding site. The humanized anti-CD 28 antibody maintained affinity for CD28 and T cell co-stimulatory activity of the 9.3mAb and was at least 40%, 50%, 75%, 80%, 90%, and in some embodiments, 100% or more effective for CD28 binding compared to the 9.3 mAb. In various embodiments, the anti-CD 28 mAb is not a superagonist.

The antibody may comprise a constant region and the constant region may be of any isotype. In some embodiments, the antibody constant region is human IgG4 or a variant thereof. In some embodiments, the constant region comprises one or more hinge stabilizing mutations, which can be introduced into the CH chain (e.g., S241, which can be substituted with P). In some embodiments, the antibody comprises a constant region, and the constant region comprises one or more mutations suitable for chemically coupling the antibody to a solid support. The one or more mutations suitable for coupling may result in an amino acid side chain functional group (e.g., thiol, amine, or hydroxyl), such as an unpaired cysteine (e.g., at S473). Other changes to the constant region include those modifications that reduce Fc γ receptor binding. For example, the CH chain may be modified at L248, e.g., L248E.

In some embodiments, the antibody is an antibody fragment, such as F (ab')₂Or Fab, or single chain antibody or other antigen-binding antibody fragment. For example, the antibody fragment can be as described hereinThe humanized mAb or other anti-CD 28 single chain variable fragment.

In some embodiments, the co-stimulatory molecule is a single chain variable fragment (scFv) comprising, or consisting essentially of, an antigen binding loop formed by the VH and VL chains of an anti-CD 28 mAb, such as an antibody described herein. The scFv antibody construct may comprise one or several (2, 3, 4 or 5) VH and VL hypervariable region chains (each chain together forming part of a 3-D epitope binding pocket) linked together in a head-to-head or head-to-tail conformation by a short peptide linker. Such constructs can be conveniently produced via a fully synthetic route due to their small size. Furthermore, scfvs may exhibit lower immunogenic potential.

In other embodiments, the co-stimulatory ligand is a bispecific construct comprising one or more HLA molecules linked to the scFv of the co-stimulatory molecule ligand or the inhibitory ligand. The antigen presenting complex and co-stimulatory or inhibitory ligand may be conjugated via a peptide tether that allows the bispecific construct to be covalently attached to the nanoparticle surface. In some embodiments, such constructs produce the same activity as larger constructs containing HLA and nanoparticles of co-stimulatory or inhibitory ligands each independently attached to the surface of the NP, thereby providing a production advantage.

The co-stimulatory molecule can be conjugated to a solid support with a complex of antigen presenting molecules to induce antigen-specific T cells. The antigen presenting molecule complex may comprise a class I and/or class II MHC complex, or a portion thereof comprising an antigen-binding groove. In some embodiments, the molecular complex comprises one or two HLA amino acid sequences, which may contain additional heterologous sequences, such as immunoglobulin sequences. Alternative heterologous sequences include dimerizing amino acid sequences such as c-fos and c-jun. In some embodiments, HLA-fusions provide additional advantages in stability and/or binding affinity.

In various embodiments, the antigen presenting complex is an MHC class I molecule complex or an MHC class II molecule complex, or alternatively CD1 d. The MHC class I molecule complex may comprise at least two fusion proteins. The first fusion protein comprises a first MHC class I alpha chain and a first immunoglobulin heavy chain, and the second fusion protein comprises a second MHC class I alpha chain and a second immunoglobulin heavy chain. The first and second immunoglobulin heavy chains associate to form an MHC class I molecule complex. The MHC class I molecule complex comprises a first MHC class I peptide binding pocket and a second MHC class I peptide binding pocket. The MHC class II molecule complex may comprise at least four fusion proteins. The two first fusion proteins comprise (i) an immunoglobulin heavy chain and (II) an extracellular domain of an MHC class II β chain. The two second fusion proteins comprise (i) an immunoglobulin light chain and (II) an extracellular domain of an MHC class II alpha chain. The two first and two second fusion proteins associate to form an MHC class II molecule complex. The extracellular domain of the MHC class II β chain of each first fusion protein and the extracellular domain of the MHC class II α chain of each second fusion protein form an MHC class II peptide binding pocket. The antigenic peptide binds to the peptide binding groove. In various embodiments, the immunoglobulin sequence is a partial heavy chain sequence comprising a hinge region to support dimerization.

In some embodiments, the antigen presenting complex is a synthetic or recombinant HLA monomer engineered to contain unpaired cysteines or to use naturally occurring unpaired cysteines for conjugation to nanoparticles. In addition, the costimulatory signal (or other antibody-based ligand) can be a Fab or scFv. In such embodiments, the two signals may be recombined in a single multifunctional construct comprising HLA molecules tethered to antigen-binding antibody fragments (e.g., scFv) that bind to the desired receptor.

In other aspects and embodiments, the invention provides bead-or particle-conjugated molecular complexes for presenting antigens to T cells, wherein the complexes comprise humanized immunoglobulin sequences or portions thereof fused to antigen presenting sequences, such as HLA amino acid sequences. In some embodiments, the immunoglobulin sequence is a human heavy chain sequence (e.g., an IGHV4 framework). The variable region does not comprise antigen binding activity against CD28 or other human proteins. The HLA amino acid sequence may be HLA-a 02:01(IMGT accession number HLA00005) or a derivative or fragment thereof, such as a derivative having from 1 to 10 or from 1 to 5 amino acid substitutions, deletions or insertions. The humanized immunoglobulin sequence may additionally comprise a linker amino acid sequence between the HLA and immunoglobulin sequences. Preferably, the linkage is devoid of immunogenicity. The molecular complex may additionally comprise a β 2 microglobulin peptide.

In various embodiments, the immunoglobulin fusion sequence is of the IgG, IgD, IgA, or IgM isotype and can be derived from any human germline framework. The germline framework includes IGHV4 (e.g., IGHV4-59), which may or may not contain one or more murine framework residues described with respect to anti-CD 28. In some embodiments, according to this aspect, the heavy chain (with or without murine framework residues) of the anti-CD 28 antibody described above is fused to HLA, and in such embodiments, the variable region is modified to reduce or eliminate CD28 binding.

In some embodiments, the HLA fusion construct does not contain a variable chain sequence. For example, the HLA or antigen presenting complex may be fused to an Ig constant region sequence above the hinge region to provide a dimeric HLA. For example, HLA or an antigen presenting portion thereof may be conjugated to the CH1 portion of each IgG heavy chain. All IgG molecules consist of two identical heavy chains (constant and variable) linked together by disulfide bonds in the hinge region (upper and lower). For example, in some embodiments, an HLA molecule or antigen presenting complex is fused to CH1 (the N-terminal end of the IgH chain above the hinge region), thereby producing a dimeric fusion protein that is smaller due to the lack of any VH and VL light chain sequences. Such constructs may provide production advantages, as well as exhibiting less immunogenic potential.

In other embodiments, the antigen presenting complex (e.g., HLA sequence) does not contain an Ig fusion partner and is a monomer. For example, in some embodiments, the C-terminal end of an antigen-presenting complex or HLA molecule (e.g., HLA-a2, etc.) contains a peptide tether sequence suitable for site-directed binding to a functional group (e.g., a maleimide moiety) on a solid/semisolid matrix such as a synthetic nanoparticle (e.g., PLGA-PEG-maleimide block copolymer, or other particles described herein). The tethering sequence may comprise any suitable sequence, which may consist essentially of hydrophilic residues such as Gly, Ser, Ala and Thr, such as two, three, four or five repeats of GGGSG or AAAGG, with cysteine residues incorporated somewhere within about 5 to about 15 (or about 5 to about 10 amino acids) tethers. The cysteine residues should be incorporated at sites which are not predicted to form intramolecular disulfide bonds.

In some embodiments, the HLA-Ig fusion or other HLA construct further comprises an HLA-binding antigenic peptide for presentation to a T cell. The antigenic peptide may comprise an antigenic portion for one or more of tyrosinase, hTERT, MAGE-1, MAGE-3, gp-100, NY-ESO-1, Melan a/Mart-1, HPV 16-E7, gp 75/brown, BAGE and S-100 and/or any of the antigenic peptides presented by class I or class II complexes as described in WO 2004/006951 (the contents of which are hereby incorporated by reference in their entirety). The HLA complexes can be attached to a solid support, such as a bead or particle as described, for presenting antigens to T cells, optionally with a costimulatory signal.

Other signals that may be provided with the antigen presenting complex include: CD80(B7-1), CD86(B7-2), B7-H3, 4-1BBL, CD27, CD30, CD134(OX-40L), B7H (B7RP-1), CD40, LIGHT, (or optionally humanized Ig fusions as described herein of such molecules or active portions thereof), antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, antibodies that specifically bind to OX40, antibodies that specifically bind to Fas, antibodies that specifically bind to PD1, antibodies that specifically bind to GITR, and antibodies that specifically bind to 4-1 BB.

The adhesion molecules for the antigen presenting platform of the present invention may mediate the adhesion of the platform to T cells or to T cell precursors. Adhesion molecules useful in the present invention include, for example, ICAM-1 and LFA-3.

T cell growth factors affect proliferation and/or differentiation of T cells. Examples of T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. Particularly useful cytokines include IL-2, IL-4, IL-7, IL-10, IL-12, IL-15 and interferon gamma. The T cell growth factor may be encapsulated in beads or particles or chemically conjugated or adsorbed to the surface. Thus, in some embodiments, the nanoparticle further comprises a therapeutic compound or protein/peptide (such as a chemotherapeutic agent, cytokine or interleukin such as IL-2, chemokine such as CCL9 that attracts T cells, and/or checkpoint inhibitory molecule such as anti-PD 1 antibody or anti-PD 1 peptide) embedded in the hydrophobic core of the particle. In some embodiments, such aapcs are constructed to target specific cells for stimulation or inhibition as well as reprogramming. In some embodiments, the embedded compound is released by degrading the particle matrix. Such aapcs can make combination therapy more tolerable and effective by limiting unwanted activity due to off-target interactions.

Antigens represented according to aspects of the invention include tumor-associated antigens. Tumor-associated antigens include unique tumor antigens expressed only by the tumor from which the antigen is derived, common tumor antigens (carcinoembryonic antigen, cancer/testis antigen) that are expressed in many but not in normal adult tissues, and tissue-specific antigens that are also expressed by normal tissues from which the tumor is derived. The tumor-associated antigen can be, for example, an embryonic antigen, an antigen with aberrant post-translational modifications, a differentiation antigen, a product of a mutated oncogene or tumor suppressor, a fusion protein, or an oncogenic viral protein. A variety of tumor-associated antigens are known in the art, and many of these are commercially available. Carcinoembryonic and embryonic antigens include cancerous embryonic antigen and alpha-fetoprotein (typically highly expressed only in developing embryos, but typically highly expressed by liver tumors and colon tumors, respectively), placental alkaline phosphatase sialyl-LewisX (expressed in adenocarcinomas), CA-125 and CA-19 (expressed in gastrointestinal, liver, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many cancers), pancreatic carcinoembryonic antigen, 5T4 (expressed in gastric cancers), alpha-fetoprotein receptor (expressed in various tumor types, particularly mammalian tumors), and M2A (expressed in germ cell neoplasias).

In some embodiments, the at least one antigen is a cancer/testis (CT) antigen, which may include NY-ESO-1, MAGE-A, B and C, CTAG-1, CTAG-45, GAGE and SSX, which are normally expressed in germ cells of the testis and not expressed in normal adult human tissue. However, many types of cancer cells have been shown to express the CT antigen, including melanoma, breast, liver, lung, ovarian, and hodgkin's lymphoma.

Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and specific surface immunoglobulins (expressed in lymphoma).

Mutant oncogene or tumor suppressor gene products include Ras and ρ 53, both of which are expressed in many tumor types, Her-2/neu (expressed in breast-and gynecological cancers), EGF-R, estrogen receptors, progesterone receptors, retinoblastoma gene products, myc (associated with lung cancer), Ras, a p53 non-mutant associated with breast cancer, MAGE-1 and MAGE-3 (associated with melanoma, lung cancer or other cancers).

Other tumor antigens include fusion proteins expressed in chronic myeloid leukemia such as BCR-ABL, and oncogenic viral proteins present in neck cancer such as HPV types 16, E6, and E7. Tissue-specific tumor antigens include melanotransferrin and MUCl (expressed in pancreatic and breast cancers); CD 10 (previously known as the common acute lymphoblastic leukemia antigen or cala) or surface immunoglobulin (expressed in B-cell leukemia and lymphoma); the alpha chain of the IL-2 receptor, T cell receptor, CD45R, CD4+/CD8+ (expressed in T cell leukemias and lymphomas); prostate specific antigen and prostatic acid phosphatase (expressed in prostate cancer); gp100, MelanA/Mart-1, tyrosinase, gp 75/brown, BAGE and S-100 (expressed in melanoma); cytokeratin (expressed in various cancers); and CD19, CD20, and CD37 (expressed in lymphomas).

In some embodiments, the antigenic peptides include MART-1, gp100, NY-ESO-1, and MAGE-A3 presented by an HLA antigen presenting complex described herein (such as an HLA-Ig fusion complex described herein).

In other embodiments, the composition comprises a mixture of multiple antigens of the tumor type, such as at least 2, 3, 4, 5, 6, 7, 8,9, or 10 antigens (e.g., 2 to 10 or 3-8 antigens).

In some embodiments, the antigen is a self-antigen, which is the organism's own "self-antigen" to which the organism generates an immune response. Autoantigens are involved in autoimmune diseases such as Goodpasture's syndrome, multiple sclerosis, Graves' disease, myasthenia gravis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris, Edison's disease, dermatitis herpetiformis, celiac disease, and Hashimoto's thyroiditis. For example, diabetes-associated autoantigens include insulin, Glutamic Acid Decarboxylase (GAD), and other islet cell autoantigens, such as ICA 512/IA-2 protein tyrosine phosphatase, ICA12, ICA69, preproinsulin or immunologically active fragments thereof (e.g., insulin B-chain, A-chain, C-peptide or immunologically active fragments thereof), IGRP, HSP60, carboxypeptidase H, peripherins, gangliosides (e.g., GM1-2, GM3) or immunologically active fragments thereof.

In some embodiments, the one or more antigens are of an infectious agent, such as protozoa, bacteria, fungi (both single and multi-cellular), viruses, prions, intracellular parasites, components of worms, and other infectious agents that can induce an immune response. Bacterial antigens include antigens of gram-positive cocci, gram-negative bacilli, gram-negative bacteria, anaerobic bacteria, such as organisms of the following families: organisms of the families Actinomycetaceae, Bacillaceae, Bartonellaceae, Bordetella, Captophagaceae, Corynebacteriaceae, Enterobacteriaceae, Legioneaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pasteurellaceae, Pseudomonadaceae, spirochaetaceae, Vibrionaceae and the genera: acinetobacter, Brucella, Campylobacter, Erwinia, Everwena, Francisella, GardnerellaHelicobacter, listeria, streptomyces, and malabsorptive genera. Antigens of protozoan infectious agents include malaria parasites, Leishmania species, Trypanosoma species and Hematophaga speciesAn antigen of a species. Fungal antigens include antigens of the genera Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides brasiliensis (Paracoccus), Sporothrix, organisms of the order Mucor, organisms inducing chromomycosis and bytylomycosis, and organisms of the genera Trichophyton, Microsporum, Epidermophyton and Malassezia. Prion antigens include the sialoglycoprotein PrP 27-30 of prions that cause scrapie, Bovine Spongiform Encephalopathy (BSE), feline spongiform encephalopathy, kuru, Kruezfeldt-Jakob Disease (CJD), Gerstmann-Strassler-Scheinker Disease (GSS), and Fatal Familial Insomnia (FFI). Intracellular parasites from which antigenic peptides can be obtained include, but are not limited to, organisms of the Chlamydiaceae, Mycoplasmaceae, acholeraceae, rickettsia, and both the coxsaceae and Elekischia genera. Viral peptide antigens include, but are not limited to, those of adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus, poxvirus, HIV, influenza virus, and CMV. Particularly useful viral peptide antigens include HIV proteins such as HIV gag protein (including but not limited to Membrane Anchor (MA) protein, nucleocapsid (CA) protein, and Nucleocapsid (NC) protein), HJV polymerase, influenza matrix (M) protein, and influenza Nucleocapsid (NP) protein, hepatitis b surface antigen (HBsAg), hepatitis b core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis b DNA polymerase, hepatitis c antigen, and the like.

Antigens, including antigenic peptides, can be actively or passively bound to the antigen binding groove of an antigen presenting complex, as described in U.S. Pat. No.6,268,411, which is hereby incorporated by reference in its entirety. Optionally, the antigenic peptide may be covalently bound to the peptide binding groove.

If desired, a peptide tether may be used to attach the antigenic peptide to the peptide-binding groove. For example, crystallographic analysis of a variety of class I MHC molecules showed that the amino terminus of β 2M is very close to the carboxy terminus of the antigenic peptide residing in the MHC peptide binding pocket, about 20.5 angstroms apart. Thus, using a relatively short linker sequence, about 13 amino acids in length, one can tether the peptide to the amino terminus of β 2M. If the sequence is appropriate, the peptide will bind to the MHC binding groove (see U.S. Pat. No.6,268,411).

The antibody or fragment and/or antigen presenting complex may be conjugated to a solid support for ex vivo or in vivo antigen presentation. Various solid supports are described in WO 2004/006951, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the solid support is a bead or particle having a functional group for coupling a ligand. The material may be a biodegradable organic material such as cellulose or dextran. In some embodiments, block copolymer trafficking (traffic) is selected to specific structural sites and biodegrades at specific intervals, i.e., with longer or shorter plasma half-life, or longer or shorter tissue residence time.

In some embodiments, the beads or particles comprise a polymer, such as one or more of the following: cyclodextrin-containing polymers, cationic-containing cyclodextrin polymers, poly (D, L-lactic-co-glycolic acid) (PLGA), poly (caprolactone) (PCL), ethylene-vinyl acetate polymers (EVA), poly (lactic acid) (PLA), poly (L-lactic acid) (PLLA), poly (glycolic acid) (PGA), poly (L-lactic-co-glycolic acid) (PLLGA), poly (D, L-lactide) (PDLA), poly (L-lactide) (PLLA), PLGA-b-poly (ethylene glycol) -PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-maleimide (PLGA-PEG-mal), poly (D, L-lactide-co-caprolactone-glycolide), poly (D, L-lactide-co-PEO-co-D, L-lactide), poly (D, L-lactide-co-PPO-co-D, L-lactide), polyalkylcyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly (hydroxy acid), polyanhydrides, polyorthoesters, poly (ester amides), polyamides, poly (ester ethers), polycarbonates, polyolefins such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly (ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate), polyvinyl halides such as poly (vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, Polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acid such as poly (methyl (meth) acrylate) (PMMA), poly (ethyl (meth) acrylate), poly (butyl (meth) acrylate), poly (isobutyl (meth) acrylate), poly (hexyl (meth) acrylate), poly (isodecyl (meth) acrylate), poly (lauryl (meth) acrylate), poly (phenyl (meth) acrylate), poly (methyl (acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl (acrylate) (polyacrylic acid), and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polytrimethylene fumarate), polyoxymethylene, poloxamers, poly (ortho esters), poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes and polyphosphoesters, and blends and/or block copolymers of two or more such polymers. Other pharmaceutically acceptable materials, such as latex, may also be used as a solid particle support.

In some embodiments, the antigen presenting complex and the co-stimulatory signal are conjugated to PLGA or PLGA-PEG particles having surface functional groups on the terminal end of the polymer (e.g., the end facing away from the surface of the particle), such as PLGA-PEG-maleimide particles (which provide functional groups on the hydrophilic outer surface for chemical coupling). In some embodiments, aapcs persist in peripheral blood circulation for a sufficient time to allow distribution to target tissues, including transport to lymph nodes, via blood/lymph exchange. The composition of the shell may also affect biodistribution. Thus, in various embodiments, the particles have a hydrophilic shell, which can be achieved by PEG of a PLGA-PEG copolymer. In various embodiments, the particles have a slightly negative charge, for example due to the combination of COOH groups on PLGA and the charge caused by targeting ligands attached to the particle surface. In some embodiments, the particles (with or without conjugated ligands) have a surface charge of about 0 to about-20 mV, or in some embodiments, -5 to-15 mV, or about-10 mV.

Nanoparticles comprising PLGA-PEG copolymers are described, for example, in U.S. patent 8,420,123, which is incorporated herein by reference.

The particles may differ in shape from irregular to spherical and/or have an uneven or irregular surface from having a smooth surface. Spherical particles have a smaller surface area than particles of irregular size. If spherical particles are used, less reagent is necessary due to the reduced surface area. In other words, the irregularly shaped particles have a significantly larger surface area than spherical particles, which provides the cells with conjugated protein content per surface area and surface area contact. For example, the asymmetric nanoparticle may have a radius of curvature of the at least one surface along the at least one axis in one of the following ranges: (a) about 1nm to about 10 nm; (b) about 11nm to about 100 nm; (c) about 101nm to about 400 nm; (d) about 401nm to about 1 μm; (e) about 10 μm to about 20 μm; (f) about 20 μm to about 100 μm; and (g) about 101 μm to about 1 mm. In some embodiments, the asymmetric nanoparticle may have an asymmetric shape defined by: a dimension (a) along the x-axis, a dimension (b) along the y-axis, and a dimension (c) along the z-axis, wherein at least one of (a), (b), or (c) is not equal to at least one other of (a), (b), or (c). In some embodiments, the asymmetric shape is an ellipsoid, which can be described by one of the following equations: a > b ═ c (prolate spheroids); a > b > c (triaxial ellipsoid); and a ═ b > c (oblate ellipsoid). Asymmetric nanoparticles that can be used according to the present invention are described in WO 2013/086500, which is hereby incorporated by reference in its entirety.

The size of the particles may vary. In various embodiments, the particle size (nominal diameter) is in the range of 0.05 to 50 μm, or in some embodiments, 0.05 to 35 μm, or in some embodiments, 0.05 to 10 μm, and in some embodiments, about 0.05 to about 3.0, about 4.0, or about 5.0 μm. For example, in some embodiments, the particle diameter or average diameter is 50 to 500 nm. In some embodiments, the particles have an average size of less than about 400nm, about 300nm, about 200nm, or about 100nm to allow better peripheral blood circulation. In some embodiments, the nanoparticles have an average size (e.g., diameter or largest axis) of about 100nm, about 150nm, or about 200 nm. The term "about," when associated with a numerical feature, means ± 10%. In some embodiments, at least 90% of the particles are in the range of about 50 to about 250nm, such as about 100 to about 150 nm. The particles may be uniform in size or may differ in size, with the average particle size preferably being as described above. In some embodiments, the particles are small enough to take advantage of the "EPR effect" (enhanced permeation and retention effects).

The ligands and molecular complexes described herein can be chemically conjugated to the beads using any useful process. Functional groups for ligand attachment include PEG-COOH, PEG-NH2, PEG-SH or other functional groups attached to different polymers such as polycyanoacrylates or polycaprolactone.

For example, the solid support may be coated before the protein binds to its surface. Once the coating chemistry has been selected, the surface of the solid support can be activated to specifically bind specific protein molecules. Thus, the coating may be selected in view of optimal reactivity and biocompatibility with various cell populations. Preferably, whatever the coating chemistry used, it provides a suitable matrix for further activation of the chemistry. A variety of such coatings are well known in the art. For example, the solid support may be coated with human serum albumin, tris (3-mercaptopropyl) -N-glycylamino) methane (U.S. patent 6,074,884), gelatin-aminodextran (U.S. patent 5,466,609), or amino acid homopolymers or random copolymers. In one embodiment, a random amino acid copolymer comprising poly (glutamic acid, lysine, tyrosine) [6:3:1] is used; this copolymer was obtained from Sigma Chemical co. under product number P8854. It is a linear random polymer of the amino acids glutamic acid, lysine and tyrosine in a ratio of 6 parts glutamic acid, 3 parts lysine and 1 part tyrosine. In another embodiment, an amino acid copolymer is used that includes lysine and tyrosine in a ratio of 4 parts lysine to 1 part tyrosine. In yet another embodiment, an amino acid copolymer is used that includes lysine and alanine in a ratio of 1 part lysine to 1 part alanine. In another embodiment, the solid support is coated with a synthetic polymer, which is then activated before it is attached to the protein molecule.

In some embodiments, the molecule is directly attached to the solid support by adsorption or by direct chemical bonding (including covalent bonding). See, e.g., Hermanson, Bloconjugate TECHNIQUES, Academic Press, New York, 1996. The molecule itself can be directly activated to have multiple chemical functional groups, including nucleophilic, leaving, or electrophilic groups. Activating functional groups include alkyl and acyl halides, amines, sulfhydryl groups, aldehydes, unsaturated bonds, hydrazides, isocyanato groups, isothiocyanates, ketones, and other groups known to activate for chemical bonding. Alternatively, the molecule may be bound to the solid support by using a small molecule-coupling reagent. Non-limiting examples of coupling reagents include carbodiimides, maleimides, N-hydroxysuccinimide esters, bis-chloroethylamines, bifunctional aldehydes such as glutaraldehyde, anhydrides, and the like. In other embodiments, the molecule may be coupled to the solid support by affinity binding such as biotin-streptavidin linkage or coupling, as is well known in the art. For example, streptavidin may be bound to a solid support via covalent or non-covalent linkage, and the biotinylated molecule may be synthesized using methods well known in the art.

In some embodiments, the particles or beads are polymers, such as PLGA-PEG, PLGA-PEG-maleimide, or ester-entrapped PLGA, wherein functional groups for surface ligand conjugation are generated during polymerization. The maleimide groups provide the formed particles with a hydrophilic "stealth" coating (PEG) on the outer surface of the particles, and functional groups attached to the shell that can be used to covalently attach ligands having at least one available free sulfhydryl (-SH) group. For example, HLA-Ig ligands and/or anti-CD 28 may be constructed on a human IgG4 framework (as described herein) containing S473C substitutions in the Fc. The unpaired cysteine residue at 473 of HLA-Ig or anti-CD 28 can be conjugated to a PEG-linked maleimide group under mild reducing conditions. Mild reducing conditions are unlikely to irreversibly denature the protein, particularly the HLA- β -2-microglobulin portion of HLA-Ig molecules.

In one exemplary embodiment, the nanoparticles have a core (PLGA) that may be prone to a particular rate of in vivo biodegradation (by adjusting the LA: GA ratio and/or mw of the PLGA polymer), a hydrophilic shell that protects it from serum protein conditioning and removal from circulation (e.g., a "PEG brush") in a range of about 50nm up to about 5 μm (e.g., average diameter or maximum axis), a surface functionalized with a uniformly controlled ligand density (1 molecule/maleimide group random relationship), and ligands facing away from the core. In some embodiments, the LA: GA ratio is from 60%/40% to 40%/60%, and in some embodiments, is about 50%/50%. In some embodiments, the PLGA has a molecular weight of about 25K to about 35K (e.g., about 30K), and the PEG has a molecular weight of about 3K to about 10K, such as about 5K. In some embodiments, the core particle has a diameter of about 150nm or about 200 nm.

In an alternative embodiment, the support may be coated with a polymer containing one or more chemical moieties or functional groups that are useful for covalent attachment to a suitable reactant, typically through a linker.

The activation chemistry allows for specific, stable attachment of molecules to the surface of the solid support. There are a variety of methods available for attaching functional groups to proteins. For example, the common cross-linking agent glutaraldehyde can be used to link the protein amine groups to the aminated solid support code in a two-step process. The resulting bond is stable to hydrolysis. Other methods include the use of crosslinkers containing n-hydroxy-succinimidyl (NHS) esters that react with amines on proteins, crosslinkers containing active halogens that react with amine-, sulfhydryl-, or histidine-containing proteins, crosslinkers containing epoxides that react with amines or sulfhydryl groups, conjugation between maleimide and sulfhydryl groups, and oxidation of pendant sugar moieties by periodate followed by reductive amination to form protein aldehyde groups.

Attachment of a particular protein to the surface of a solid support can be achieved by direct coupling of the protein or by using an indirect method. When coupled to a linker or spacer protein such as anti-mouse IgG or streptavidin, some proteins will have them directly linked or conjugated while others retain better functional activity. If desired, a linker or a connexin may be used. Ligands such as the antigen presenting complex and co-stimulatory molecule may be modified with amino acid substitutions to allow chemical conjugation.

The ratio of specific proteins on the same solid support may be varied to increase the effectiveness of the solid support in antigen or antibody presentation. For example, the ratio of antigen presenting complex to anti-CD 28 may be at least about 30:1, or at least about 10:1, about 3:1, about 1:1, about 0.3: 1; about 0.1:1, and at least about 0.03: 1. In some embodiments, the ratio is about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1, about 1:2, about 1:3, about 1:4, or about 1: 5. The total amount of protein coupled to the support may be at least 10mg/ml, at least 25mg/ml, at least 50mg/ml, at least 100mg/ml, at least 150mg/ml, or greater than 200 mg/ml. In some embodiments, such as those employing PLGA or PLGA-PEG particles having surface functional groups (e.g., maleimide or ester), the total amount of protein coupled to the particles may be 1 to 10 μ g/mg PLGA, or in some embodiments, 2 to 6 μ g/mg PLGA. In some embodiments, the particle has a ligand density of about 10³To about 10⁵Ligand/. mu.m²Or in some embodiments about 10⁴Ligand/. mu.m². For example, for nanoparticles having a size in the range of 100 to 200nm, the nanoparticles have on average about 100 to about 1500 ligands, such as about 200 to about 1200 ligands, or about 400 to about 1000 ligands, or about 500 to about 800 ligands.

In various embodiments, the present invention provides pharmaceutical compositions comprising polymeric beads or particles, anti-CD 28 antibodies as described herein, and/or antigen presenting complexes such as humanized Ig HLA fusion complexes as described herein. The pharmaceutical composition may additionally comprise an antigenic peptide for presentation to T cells as described and may be co-formulated with conjugated beads or particles. In various embodiments, the pharmaceutical composition is shelf-stable, and in some embodiments, is provided in a lyophilized form for reconstitution prior to administration, or in another "off-the-shelf" pharmaceutical formulation.

In some embodiments, the present invention provides pharmaceutical compositions comprising PLGA-or PLGA-PEG-based nanoparticles having a diameter or average diameter of 50 to 500nm and comprising a surface-conjugated anti-CD 28 antibody and an antigen presenting complex. The anti-CD 28 antibody can be a humanized antibody, such as a humanized antibody as described herein, and can be an antibody fragment such as a single chain variable fragment. In some embodiments, the antigen presenting complex comprises at least one HLA antigen-binding groove. The anti-CD 28 and HLA complex can be coupled to the particle separately or together in the same reaction. The pharmaceutical composition may comprise at least one peptide antigen, such as a tumor antigen (e.g., MART-1), and it may be co-formulated with the particles without any active loading process.

The pharmaceutical compositions described herein can be used in immunotherapy, for example in a method for inducing antigen-specific cytotoxic T cell formation, by administering an effective amount of the composition to a patient in need thereof. In some embodiments, the patient is a cancer patient.

The particle-based antigen presentation platforms described herein may be administered to a patient by any suitable route, including intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intralymphatic administration, and intratumoral administration. Patients include human and veterinary patients.

Some exemplary embodiments of the invention are described below.

In some embodiments, the present invention provides pharmaceutical compositions comprising polymeric PLGA-PEG particles having a size in the range of about 100 to 200nm, a surface charge of about-0 to-20 mV (and in some embodiments-5 to-15 mV), and about 100 to 1500 protein ligands per particle. In some embodiments, the protein ligands are each coupled to the particle by sulfhydryl-maleimide chemistry. The ligands include anti-CD 28 antibody ligand populations and HLA ligand populations and one or more antigenic peptides for presentation to T cells. The composition comprises a pharmaceutically acceptable carrier for intravenous, intraarterial, subcutaneous, intradermal, intralymphatic or intratumoral administration.

In some embodiments, the particles are substantially spherical or approximately spherical.

In some embodiments, the PLGA is a polymer of about 50% Lactic Acid (LA) and 50% Glycolic Acid (GA).

In some embodiments, the PLGA polymer has a molecular weight of about 30K, and the PEG has a molecular weight of about 3K to about 10K, such as about 5K.

In some embodiments, the composition has 400 to 1000 ligands per particle.

In some embodiments, the anti-CD 28 antibody ligand comprises a human IGHV4-59 germline framework optionally having 5 to 15 murine framework residues, and an IGKV4-01 germline framework optionally having 3 to 15 murine framework residues.

In some embodiments, the anti-CD 28 is an ScFv.

In some embodiments, the HLA is HLA-a 02:01, which may comprise a fusion to an immunoglobulin sequence above the hinge region sufficient to provide a dimeric HLA construct.

In some embodiments, the composition is lyophilized.

In particular, the antigen presentation platform may be used to treat patients with infectious diseases, cancer or autoimmune diseases, or to provide prophylactic protection to immunosuppressed patients.

Infectious diseases that may be treated include those caused by bacteria, viruses, prions, fungi, parasites, worms, and the like. Such diseases include AIDS, hepatitis, CMV infection and post-transplant lymphoproliferative disease (PTLD). CMV, for example, is the most common viral pathogen present in organ transplant patients, and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation (Zaia, hematol. oncol. clin. north am.4,603-23,1990). This is due to an immunocompromised condition in these patients, which results in reactivation of latent viruses in seropositive patients or opportunistic infections in seronegative individuals. Current treatment focuses on the use of antiviral compounds such as ganciclovir, which have disadvantages, the most notable of which is the emergence of drug resistant CMV. One useful alternative to these treatments is a prophylactic immunotherapy regimen, which involves the generation of virus-specific CTLs derived from the patient or an appropriate donor prior to initiating the transplantation procedure.

PTLD occurs in a large proportion of transplant patients and is caused by Epstein-Barr virus (EBV) infection. EBV infection is believed to be present in about 90% of the U.S. adult population (Angstromopoulos & Hummel, Histopathlogy 29,291-2)15,1996). Active viral replication and infection is prevented by the immune system, but as in the case of CMV, immunocompromised individuals lose the control T cell population as a result of transplantation therapy, which reactivates the virus. This represents a serious impediment to the migration protocol. EBV may also be involved in tumor promotion in a variety of hematologic and non-hematologic cancers. There is also a strong correlation between EBV and nasopharyngeal carcinoma. Thus, prophylactic treatment using EBV-specific T cells provides a superior alternative to current therapies.

Cancers that may be tumorous according to the invention include melanoma, cancers such as colon cancer, head and neck cancer, duodenal cancer, prostate cancer, breast cancer, lung cancer, ovarian cancer, ductal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, stomach cancer, dysplastic oral mucosa cancer, polypus cancer, invasive oral cancer, non-small cell lung cancer, transitional and squamous cell urinary cancer, and the like; neurological malignancies such as neuroblastoma, glioma, and the like; hematologic malignancies such as chronic myelogenous leukemia, childhood acute leukemia, non-hodgkin's lymphoma, chronic lymphocytic leukemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell-rich cutaneous lymphoproliferation, bullous pemphigoid, discoid lupus erythematosus, lichen planus, and the like; and the like. See, e.g., Mackensen et al, int.J. cancer 86,385-92, 2000; jonuleit et al, int.J. cancer 93,243-51, 2001; lan et al, J.immunotherpy 24,66-78,2001; meidenbauer et al, J.Immunol.170(4),2161-69, 2003.

In some embodiments, the present invention provides methods of treating cancer (including those identified above) by administering a pharmaceutical composition described herein to activate T cells having anti-tumor activity. In some embodiments, the therapy is provided with one or more immune checkpoint inhibitors such as Nivolumab (Nivolumab), Pembrolizumab, and Ipilimumab (Ipilimumab). In some embodiments, the additional therapy is anti-CTLA 4 or anti-PD 1 or anti-PD-L1. The additional therapies or checkpoint inhibition may be administered separately by their conventional regimen, or may be administered as nanoparticles described herein or additional ligands linked to a separate population of nanoparticles. In some embodiments, one or more immune checkpoint inhibitors are provided as an initial therapy, and a therapy using the nanoparticles described herein started subsequently, e.g., after about 1 to about 8 weeks of checkpoint inhibition therapy or after about 2 to about 4 weeks of checkpoint inhibition therapy. In some embodiments, the one or more checkpoint inhibitors are provided concurrently with nanoparticle therapy, e.g., at the start of therapy and about every two weeks, or at the start of therapy and about every two weeks for one or more checkpoint inhibitors and about every four weeks for nanoparticle therapy. In some embodiments, the patient is resistant to, or shows only a partial or transient response to, checkpoint inhibition therapy, and the aapcs described herein enhance tumor regression in these patients. In other embodiments, for cancers that are generally resistant to checkpoint inhibition therapy, the compositions described herein expand the success of using checkpoint inhibitors against such cancers.

In some embodiments, the peptide antigen is selected based on a personalized basis for the patient, based on an analysis of the patient's tumor. For example, the methods described in Ionov Y., A high throughput method for identifying a qualified stereo-associated antigens, Oncotarget 1(2), 148-. In these embodiments, the nanoparticles may be provided (on an "off-the-shelf basis") and tumor antigens selected and loaded on a personalized basis.

Autoimmune diseases that can be treated include asthma, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, psoriasis, myasthenia gravis, goodpasture's syndrome, graves ' disease, pemphigus vulgaris, edison's disease, dermatitis herpetiformis, celiac disease, and hashimoto's thyroiditis.

Antigen-specific helper T cells can be used to activate macrophages or activate B cells to produce specific antibodies that can be used, for example, to treat infectious diseases and cancer. Antibody-producing B cells themselves may also be used for this purpose.

The invention also provides polynucleotides encoding the amino acid sequences described herein and host cells expressing the amino acid sequences described herein.

The invention is further illustrated by the following non-limiting examples.

Examples

Example 1: design of germline humanized variable and human constant region sequences

This example demonstrates, among other things, the sequence design of germline humanized (grafted CDR) antibodies from a mouse anti-CD 28 antibody template; design of human constant region sequences (including human IgG4 containing S241P (Kabat numbering) hinge stabilization mutations, L248E (Kabat numbering) mutations to remove residual Fc γ receptor binding, and Cys residues suitable for coupling to antibodies (S473C, Kabat numbering); design of variant germline humanized antibody V domains that potentially do not bind to CD 28; design of linker sequence not containing the N-terminus of potential T cell epitopes to fuse HLA-a 02:01 to germline humanized antibody.

The starting anti-CD 28 antibody was the murine 9.3 monoclonal antibody (Tan et al J.Exp.Med.1993177: 165). 9.3 structural models of antibody V regions were generated using Swiss PDB and analyzed to identify amino acids in the V regions that may be essential for antibody binding properties. All residues contained within the CDRs (defined using Kabat and Chothia) along with multiple framework residues are considered to be of potential importance for binding. The VH and vk sequences of anti-CD 28 contain typical framework (Fw) residues, and the CDR1, 2 and 3 motifs are comparable to many murine antibodies.

For humanization, human IGHV4-59 germline Fw was selected as the template for the heavy chain (see IGHV3/OR16-10 selected by Tan et al J.Immunol 2002169: 1119-1125). IGKV4-01 germline Fw was selected as template for the light chain. These Fw are all 62% homologous to their corresponding murine VH and vk sequences. The murine CDRs were grafted into these fws, and different numbers of murine Fw residues were also included to generate three humanized VH variants and three humanized V κ variants (fig. 1-6).

For the heavy chain Fw, Fw1 residues 1 and 3 are believed to be important for antigen binding because they are adjacent to the binding pocket, while residue 6 is believed to affect the conformation of the beta chain supporting residues 1 and 3 and the conformation of CDR 3. These murine Fw residues are thus retained in all variants.

In Fw2, residue 37 was thought to be important for maintaining the interface between VH and vk, while residue 48 was thought to support the conformation of CDR 2; both residues are therefore retained in all variants.

In Fw3, residues 73, 76, and 78 directly contact the CDR1, while residue 71 contacts both the CDR1 and CDR 2; thus, these residues may be required for antigen binding (depending on the contribution of CDR1 and CDR 2) and thus remain in all variants. Residue 71 may sometimes indirectly affect the conformation of CDR1 by affecting the conformation of residues 71 to 78, while residues 82a and 82c may also indirectly affect the conformation of CDR 2. These residues are therefore only retained in VH 1. Residues 67 and 82 are adjacent in three-dimensional structure and interact to fill in the space that can affect the conformation of CDR2 and potentially the beta chain that supports CDR1 and 3. These residues are therefore retained in the variants VH1 and VH 2.

For light chain Fw, Fw1 residue 3 is adjacent to the binding pocket and can be directly involved in antigen binding, while residue 4 directly supports the conformation of CDR 3. Thus, these murine Fw residues remain in all variants.

In Fw2, residue 49 supports the conformation of CDR2, and is also important for the interface between the heavy and light chains, where it directly supports the conformation of the heavy chain CDR3, thus remaining in all variants.

In Fw3, residues 85 and 87 are believed to be important for the interface of the heavy and light chains, and also support the CDR3 conformation and thus remain in all variants. Residue 80 is thought to potentially have an indirect effect on the conformation of CDRs 2 and 3 and remains only in V κ 1. Residue 70 forms a salt bridge with the light chain residue R24 and thus has an important conformational impact on the vk domain. In anti-CD 28, the salt bridge is not present (because residue 70 is N rather than D) and the introduction of the interaction may be disadvantageous; however in murine the antibody N70 was glycosylated (NFS) and it would be beneficial to remove this during humanization; thus the murine N remains in vk1 and vk2, but changes to D in vk 3.

Human IgG4/κ -based constant region sequences were designed to include hinge stabilizing mutations (S241P) and mutations in the lower hinge that remove residual Fc γ receptor binding (L248E). Cysteine residues are also included near the C-terminus of Fc for chemical coupling purposes (S473C). The modified IgG4 heavy chain constant region sequence is shown in fig. 7, along with the kappa light chain constant region sequence (fig. 8).

Additional VH domains were designed to potentially not bind to CD28, and this sequence is shown in fig. 9. Analysis of the murine V region sequences indicated (to the extent of somatic mutation from the murine germline V region) that the VH might be the major contributor to CD28 binding. Thus only potentially non-binding humanized VH variants were designed. This variant did not contain any mouse Fw residues to reconstruct the correct CDR conformation, and also contained three mutations in CDRH3 at residues that might be important for binding (Y100A, Y100aA, Y100 bA).

Example 2: design of linkers for fusion of HLA-A02: 01 to humanized antibodies

Linker for fusion of HLA-A02: 01(IMGT accession HLA00005) to the N-terminus of humanized anti-CD 28 antibody by iTope^TMAnd TCED^TMConstructed and incorporated into the assay to remove potential immunogenicity.

iTope^TMThe software predicted a favorable interaction between the amino acid side chains of peptides within the open binding groove of the 34 human MHC class II alleles and the specific binding pocket (in particular the pocket positions; p1, p4, p6, p7 and p 9). These alleles represent the most common HLA-DR alleles present worldwide, without weighting those alleles that are most predominantly present in any particular ethnic group. Twenty alleles contained an 'open' p1 configuration, and fourteen contained a 'closed' configuration in which the glycine at position 83 was replaced with a valine. The position of the key binding residue viaComputer simulations were obtained to generate 9-mer peptides overlapping one amino acid across the test protein sequence. Comparison with physical MHC class II binding experiments has shown iTope^TMCan be used to successfully discriminate between peptides that bind and do not bind MHC class II molecules with high accuracy. Any restriction of MHC class II binding assays generated via computer simulation Using TCED^TMReduction of the TCED^TMContaining a source previously in EpiScreen^TMLarge database peptides of sequences screened in ex vivo T cell epitope mapping assays: (>10,000 peptides). Therefore TCED^TMCan be used to search any test sequence against unrelated antibody and protein sequences to find a correlation with actual ex vivo immunogenicity.

Use of iTope^TMLinker sequence analysis was performed with overlapping 9 mers spanning the linker sequence, which were tested against each of the 34 MHC class II alleles. Each 9 mer was scored based on potential 'hits' and interactions with MHC class II molecules. The peptide score calculated by the software was between 0 and 1. Will result in a high average binding score (at iTope)^TMIn the scoring function>0.55) and if > 50% of the MHC class II binding peptides (i.e.17 out of 34 alleles) have a high binding affinity (score)>0.6), such peptides are defined as "promiscuous high affinity" MHC class II binding peptides (which are considered to be high risk for containing CD4+ T cell epitopes). Wherein the MHC class II binding peptide accounts for more than or equal to 50% (score)>0.55 (but not most)>0.6)) is defined as a "promiscuous medium affinity" MHC class II binding peptide. Further analysis of the sequence used TCED^TMThe process is carried out. The sequences were used to query TCED by BLAST search^TMTo identify the previous EpiScreen^TMAny high sequence identity between peptides (T cell epitopes) of unrelated proteins that stimulated T cell responses in the study.

The sequence used by Schneck et al comprises two linkers, one at the N-terminus of HLA-a 02:01 for ligation to an N-terminal signal sequence, and one at the C-terminus for fusion to the anti-CD 28 VH domain (see e.g. fig. 9). For the N-terminal linker, the sequence was analyzed from signal sequence cleavage site to linker and included the first 8 amino acids of the HLA-a 02:01 mature protein. For the C-terminal linker, sequences were analyzed from the terminal 8 amino acids of the HLA-a 02:01 α 3 domain up to the first 8 amino acids of the linker sequence and anti-CD 28 VH domain.

Peptides with binding scores >0.6 (high affinity) bind to the majority (. gtoreq.17) of the MHC class II alleles (defined as promiscuous high affinity binders). A medium affinity binder with a binding score between 0.55 and 0.6 binds ≧ 17MHC class II alleles. The N-terminal linker was found to contain two promiscuous MHC class II binding sequences, one high affinity (p 1 anchor at position 2) and one medium affinity (p 1 anchor at position 4). The C-terminal linker was found to contain a promiscuous medium affinity MHC class II binding peptide with a p1 anchor at position 11.

BLAST search T-cell epitope database of epitopes (TCED)^TM) Using e.g. iTope^TMThe same sequences used in the analysis were performed to determine any homology to the previously identified epitopes. The TCED^TMFor aiming at large scale>10,000 peptides) from the peptide library of EpiScreen^TMIrrelevant sequences tested in the T cell epitope mapping assay. None of the linker sequences was found to be in TCED^TMContains any 'hits'.

iTope^TMFurther used to assess linker sequence changes to reduce their propensity to bind to MHC class II. It should be noted that the N-terminal linker may be completely removed such that the N-terminus of HLA-a x 02:01 is directly fused to the signal sequence provided in the pBFKsr vector or its native signal sequence. This will ensure that the N-terminus of the fusion protein will contain only human germline sequences and avoid the risk of T cell epitopes. The linker sequences recommended below were found to reduce MHC class II binding to local residual levels: (<5 alleles bound by any 9-mer) and provide restriction sites suitable for cloning (although both sequences would require modification of the vector):

example 3: codon optimization of sequences and expression clones

Use ofCodons were optimized, and optimized sequences were cloned for expression as shown below.

The sequence was engineered to have a PmeI restriction site, a Kozak sequence, and a signal peptide for expression in NS0 cells. Translation was initiated immediately downstream of the Kozak sequence.

The fully untranslated amino acid sequence of the HLA-IgG4HC fusion is shown in FIG. 10.

The untranslated sequence of LC3(VK3) is shown in FIG. 11.

The untranslated sequence of HC1 is shown in fig. 12.

The untranslated sequence of HC2 is shown in fig. 13.

Human β 2 microglobulin is also expressed.

Example 4: expression in NS0 cells

Based on Biacore affinity data and other considerations, HC1:: LC3 and HC2:: LC3 heavy and light chain combinations were selected as first and second mAb candidates, respectively, for StableFast-NS0 cell line development.

The final vector map of pBFksr:: HC1:: LC3 bicistronic expression vector for production of STABLENFAST-NS 0 cell line is depicted in FIG. 14. The construction of pBFksr:: HC2:: LC3 was performed using the same method.

Parental NS0 cells were expanded in supplemented serum-free growth medium. After establishing a healthy culture, ten million cells (10X 10)⁶) Transfection with 45. mu.g of linear (. DELTA.PvuI) expression vector DNA. Cells were allowed to recover in bulk in growth medium for 24 hours. After recovery, cells were washed in supplemented serum-free selection medium (cholesterol-), resuspended in selection medium and distributed to 40 × 96-well plates at 200 μ L per well. For HC1:: LC3 and HC2:: LC3, the actual distribution was 1140 cells/well and 840 cells/well, respectively. Plates were incubated at 37 ℃ for 1 week with 5% CO2 and fed with phenol red supplemented with selection medium. Two weeks after transfection, the medium color changed from red to red based onTurning yellow, actively growing multiple pores.

A total of 1,127 wells from HC 1:LC 3 transfections were screened for human IgG expression by ELISA. A total of 612 wells from HC 2:LC 3 transfection were screened. Based on IgG concentrations, a total of 290 and 101 cell lines were expanded to 24-well plates for HC1:: LC3 and HC2:: LC3, respectively. The 24 hour yield assay was used to select the best expressors for further analysis. Briefly, 24-well plates were prepared at 5X 10⁵Individual cells were seeded in 500 μ L fresh medium. After 24 hours, the supernatants were screened by ELISA. Based on IgG concentrations, a total of 60 and 24 cell lines were expanded to 6-well plates for HC1:: LC3 and HC2:: LC3, respectively.

A 3-day specific yield assay was used to select the best expressors for further analysis. Briefly, 6-well plates were seeded with 4 × 105 cells in 1.5mL fresh medium. After 3 days, cells were counted and supernatants were screened by ELISA. Based on IgG concentration and growth, the average specific yield or SPR in pg/cell/day can be calculated. A total of 20 and 10 cell lines were expanded to T-75 flasks for HC1:: LC3 and HC2:: LC3, respectively, based on relative SPR. The SPR assay was repeated on the T-75 scale for 3 days to select the final cell line for suspension adaptation and scale-up for mAb production.

Five cell lines for each mAb were scaled up to 30-mL shake medium and SPR and growth re-evaluated. All suspension systems were stored (banked). Cell lines that performed best for each mAb were scaled up to spinner flask media for small-scale production.

For HLA-IgG4 fusion proteins, pBFksr: HLA-IgG4: LC3 bicistronic expression vector was constructed for STABLENFAST-NS 0 cell line production. The vector is illustrated in FIG. 15. Expression cassettes and vectors containing the human β 2 microglobulin gene were also created for a tricistronic expression vector encoding all three fusion protein subunits (human HLA-IgG4 heavy chain fusion, a-CD28 light chain [ LC3], and human β 2 microglobulin). The tricaistronic construct is shown in figure 16. Expression of all three genes was confirmed in transient HEK293 cultures by ELISA and western blot analysis of the supernatants.

Example 5: functional characterization

Humanized monoclonal antibodies against CD28 were tested for their ability to induce the amplification of freshly isolated PBMCs on mAb-coated plates. As shown in fig. 17, humanized anti-CD 28 is not a superagonist.

Humanized monoclonal antibodies were tested for their ability to stain CD28 on a human T-cell line. The results are shown in FIG. 18. Fig. 18(a) shows staining with murine anti-human CD8 mAb (clone 9.3, isotype IgG2 a). Black ═ unstained cells, red ═ anti-IgG 2a FITC, blue ═ anti-CD 28+ anti-IgG 2a FITC. FIG. 18(B) shows staining with humanized anti-CD 28 (isotype IgG 4). Black ═ unstained cells, red ═ anti-IgG 4 PE, blue ═ anti-CD 28(35ng) + anti-IgG 4 PE, violet ═ anti-CD 28(1 μ g) + anti-IgG 4 PE. Staining with humanized anti-CD 28 was blocked with clone 9.3mAb (not shown).

After purification of HLA-Ig, antigen peptide loading efficiency was checked by ELISA using conformation dependent anti-HLA mAb to capture peptide loaded proteins (as described in current protocol in chapter 17.2 Immunology). Reproducible loading efficiencies (i.e., correct MHC restriction) for specific peptides are expected to be-90% compared to 0% for non-specific peptides (i.e., MHC mismatch).

Is incorporated by reference

All patents and publications referenced herein are hereby incorporated by reference in their entirety.

Sequence listing

<110> Nick West Munrong Mass

<120> compositions and methods for immunotherapy

<130> FCT150900-99-DIV

<150> US 61/838,547

<151> 2013-06-24

<150> US 61/948,916

<151> 2014-03-06

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (humanized variable heavy sequence of anti-CD 28)

<220>

<221> CDS

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gag gtg aag ctg cag cag tca gga cct ggc ctg gtg aag ccc tca gag 48

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

acc ctg tcc ctc act tgt act gtc tct ggg ttt tca tta agc gac tat 96

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

ggt gtt cat tgg gtt cgc cag gct cca gga aag gga ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

gga gta ata tgg gct ggt gga ggc acg aat tat aat tcg gct ctc atg 192

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

tcc aga aag acc atc agc aaa gac aac tcc aag agc caa gtt ttc tta 240

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

aaa atg aac agt ctg aca gct gct gac aca gcc gtg tat tac tgt gcc 288

Lys Met Asn Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

aga gat aag gga tac tcc tat tac tat tct atg gac tac tgg ggc caa 336

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

ggc acc ctg gtc acc gtc tcc tca 360

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

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<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 2

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

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<213> Artificial sequence

<220>

<223> synthetic sequence (anti-CD 28 VH2)

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<221> CDS

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gag gtg aag ctg cag cag tca gga cct ggc ctg gtg aag ccc tca gag 48

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

acc ctg tcc ctc act tgt act gtc tct ggg ttt tca tta agc gac tat 96

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

ggt gtt cat tgg gtt cgc cag gct cca gga aag gga ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

gga gta ata tgg gct ggt gga ggc acg aat tat aat tcg gct ctc atg 192

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

tcc aga aag acc atc agc aaa gac aac tcc aag agc caa gtt tcc tta 240

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

aaa atg agc agt gtg aca gct gct gac aca gcc gtg tat tac tgt gcc 288

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

aga gat aag gga tac tcc tat tac tat tct atg gac tac tgg ggc caa 336

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

ggc acc ctg gtc acc gtc tcc tca 360

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 4

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 5

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<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (anti-CD 28 VH3)

<220>

<221> CDS

<222> (1)..(360)

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gag gtg aag ctg cag cag tca gga cct ggc ctg gtg aag ccc tca gag 48

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

acc ctg tcc ctc act tgt act gtc tct ggg ttt tca tta agc gac tat 96

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

ggt gtt cat tgg gtt cgc cag gct cca gga aag gga ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

gga gta ata tgg gct ggt gga ggc acg aat tat aat tcg gct ctc atg 192

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

tcc aga gtg acc atc agc aaa gac aac tcc aag agc caa gtt tcc tta 240

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

aaa ctg agc agt gtg aca gct gct gac aca gcc gtg tat tac tgt gcc 288

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

aga gat aag gga tac tcc tat tac tat tct atg gac tac tgg ggc caa 336

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

ggc acc ctg gtc acc gtc tcc tca 360

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 6

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 6

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of sequences (anti-CD 28 VK1)

<220>

<221> CDS

<222> (1)..(333)

<400> 7

gac atc gag ctc act cag tct cca gat tct ttg gct gtg tct cta ggg 48

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

gag aga gcc acc atc aac tgc aga gcc agt gag agt gtt gaa tat tat 96

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

gtc aca agt tta atg cag tgg tac cag cag aag cca gga cag cca ccc 144

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

aaa ctc ctc atc ttt gct gca tcc aac gta gaa tct ggg gtc cct gac 192

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

agg ttt agt ggc agt ggg tct ggg aca aac ttc acc ctc acc atc tct 240

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Thr Ile Ser

65 70 75 80

tct ctg cag gag gag gat gtt gca atg tat ttc tgt cag caa agt agg 288

Ser Leu Gln Glu Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

aag gtt cct tac acg ttc gga ggg ggg acc aag gtg gaa ata aaa 333

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 8

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 8

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Glu Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 9

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of sequences (anti-CD 28 VK2)

<220>

<221> CDS

<222> (1)..(333)

<400> 9

gac atc gag ctc act cag tct cca gat tct ttg gct gtg tct cta ggg 48

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

gag aga gcc acc atc aac tgc aga gcc agt gag agt gtt gaa tat tat 96

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

gtc aca agt tta atg cag tgg tac cag cag aag cca gga cag cca ccc 144

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

aaa ctc ctc atc ttt gct gca tcc aac gta gaa tct ggg gtc cct gac 192

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

agg ttt agt ggc agt ggg tct ggg aca aac ttc acc ctc acc atc tct 240

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Thr Ile Ser

65 70 75 80

tct ctg cag gcc gag gat gtt gca atg tat ttc tgt cag caa agt agg 288

Ser Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

aag gtt cct tac acg ttc gga ggg ggg acc aag gtg gaa ata aaa 333

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 10

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 10

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 11

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of sequences (anti-CD 28 VK3)

<220>

<221> CDS

<222> (1)..(333)

<400> 11

gac atc gag ctc act cag tct cca gat tct ttg gct gtg tct cta ggg 48

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

gag aga gcc acc atc aac tgc aga gcc agt gag agt gtt gaa tat tat 96

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

gtc aca agt tta atg cag tgg tac cag cag aag cca gga cag cca ccc 144

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

aaa ctc ctc atc ttt gct gca tcc aac gta gaa tct ggg gtc cct gac 192

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

agg ttt agt ggc agt ggg tct ggg aca gac ttc acc ctc acc atc tct 240

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

tct ctg cag gcc gag gat gtt gca atg tat ttc tgt cag caa agt agg 288

Ser Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

aag gtt cct tac acg ttc gga ggg ggg acc aag gtg gaa ata aaa 333

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 12

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 12

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 13

<211> 984

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (modified constant heavy sequence)

<220>

<221> CDS

<222> (1)..(984)

<400> 13

gct tcc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 48

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 96

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

tac acc tgc aat gta gat cac aag ccc agc aac acc aag gtg gac aag 288

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

aga gtt gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca cct 336

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

gag ttc gag ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 384

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 432

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 480

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 528

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 576

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 624

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 672

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 720

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 768

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 816

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 864

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 912

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 960

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

ctc tgc ctg tct ctg ggt aaa tga 984

Leu Cys Leu Ser Leu Gly Lys

325

<210> 14

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Cys Leu Ser Leu Gly Lys

325

<210> 15

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (constant k light sequence)

<220>

<221> CDS

<222> (1)..(324)

<400> 15

cga act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag 48

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc 96

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 144

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc 192

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag 240

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg 288

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

ccc gtc aca aag agc ttc aac agg gga gag tgt tag 324

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 16

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 16

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 17

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (humanized non-CD 28-binding variable region)

<220>

<221> CDS

<222> (1)..(360)

<400> 17

gag gtg aag ctg cag cag tca gga cct ggc ctg gtg aag ccc tca gag 48

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

acc ctg tcc ctc act tgt act gtc tct ggg ttt aca ttc agc gac tat 96

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

ggt gtt cat tgg att cgc cag cct cca gga aag gga ctg gag tgg atc 144

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

gga gta ata tgg gct ggt gga ggc acg aat tat aat tcg gct ctc atg 192

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

tcc aga gtg acc atc agc gtg gac acc tcc aag aac caa ttt tcc tta 240

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

aaa ctg agc agt gtg aca gct gct gac aca gcc gtg tat tac tgt gcc 288

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

aga gat aag gga tac tcc gct gcc gct tct atg gac tac tgg ggc caa 336

Arg Asp Lys Gly Tyr Ser Ala Ala Ala Ser Met Asp Tyr Trp Gly Gln

100 105 110

ggc acc ctg gtc acc gtc tcc tca 360

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 18

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Ala Ala Ala Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 733

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence (humanized HLA-IgG4HC)

<400> 19

Gln Val Gln Leu Thr Arg Glu Gly Ser Gly Ser His Ser Met Arg Tyr

1 5 10 15

Phe Phe Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile

20 25 30

Ala Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp

35 40 45

Ala Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu

50 55 60

Gly Pro Glu Tyr Trp Asp Gly Glu Thr Arg Lys Val Lys Ala His Ser

65 70 75 80

Gln Thr His Arg Val Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln

85 90 95

Ser Glu Ala Gly Ser His Thr Val Gln Arg Met Tyr Gly Cys Asp Val

100 105 110

Gly Ser Asp Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp

115 120 125

Gly Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala

130 135 140

Ala Asp Met Ala Ala Gln Thr Thr Lys His Lys Trp Glu Ala Ala His

145 150 155 160

Val Ala Glu Gln Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp

165 170 175

Leu Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp

180 185 190

Ala Pro Lys Thr His Met Thr His His Ala Val Ser Asp His Glu Ala

195 200 205

Thr Leu Arg Cys Trp Ala Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu

210 215 220

Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val

225 230 235 240

Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val

245 250 255

Val Val Pro Ser Gly Gln Glu Gln Arg Tyr Thr Cys His Val Gln His

260 265 270

Glu Gly Leu Pro Lys Pro Leu Thr Trp Ala Arg Glu Val Ser Glu Val

275 280 285

Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu

290 295 300

Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser Asp Tyr Gly Val

305 310 315 320

His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val

325 330 335

Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg

340 345 350

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

355 360 365

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

370 375 380

Lys Gly Tyr Ser Ala Ala Ala Ser Met Asp Tyr Trp Gly Gln Gly Thr

385 390 395 400

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

405 410 415

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

420 425 430

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

435 440 445

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

450 455 460

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

465 470 475 480

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

485 490 495

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

500 505 510

Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu

515 520 525

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

530 535 540

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

545 550 555 560

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

565 570 575

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

580 585 590

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

595 600 605

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

610 615 620

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

625 630 635 640

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

645 650 655

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

660 665 670

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

675 680 685

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

690 695 700

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

705 710 715 720

His Tyr Thr Gln Lys Ser Leu Cys Leu Ser Leu Gly Lys

725 730

<210> 20

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence (light chain 3(LC3 or Vk3))

<400> 20

Asp Ile Glu Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence (heavy chain 1(HC1))

<400> 21

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Cys Leu Ser Leu Gly Lys

435 440 445

<210> 22

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence (heavy chain 2(HC2))

<400> 22

Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Lys Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Cys Leu Ser Leu Gly Lys

435 440 445

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 23

Asp Tyr Gly Val His

1 5

<210> 24

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 24

Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 25

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 25

Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr

1 5 10

<210> 26

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 26

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gln

1 5 10 15

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 27

Ala Ala Ser Asn Val Glu Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 28

Gln Gln Ser Arg Lys Val Pro Tyr Thr

1 5

<210> 29

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 29

Gly Gly Gly Ser Gly

1 5

<210> 30

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic sequence

<400> 30

Ala Ala Ala Gly Gly

1 5

<210> 31

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (N-terminal linker)

<220>

<221> CDS

<222> (1)..(51)

<400> 31

cag gtc caa ctg acg cgt gag ggg tcc ggc tct cac tcc atg agg tat 48

Gln Val Gln Leu Thr Arg Glu Gly Ser Gly Ser His Ser Met Arg Tyr

1 5 10 15

ttc 51

Phe

<210> 32

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 32

Gln Val Gln Leu Thr Arg Glu Gly Ser Gly Ser His Ser Met Arg Tyr

1 5 10 15

Phe

<210> 33

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic sequence (C-terminal linker)

<220>

<221> CDS

<222> (1)..(57)

<400> 33

gag ggt ttg ccc aag ccc ctc acc tgg gct cga gag gtg agc gag gtc 48

Glu Gly Leu Pro Lys Pro Leu Thr Trp Ala Arg Glu Val Ser Glu Val

1 5 10 15

aag ctg cag 57

Lys Leu Gln

<210> 34

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 34

Glu Gly Leu Pro Lys Pro Leu Thr Trp Ala Arg Glu Val Ser Glu Val

1 5 10 15

Lys Leu Gln