B-cell receptor complex binding proteins containing T-cell epitopes

Abstract

The present invention relates to a polypeptide comprising a) a binding peptide binding to at least one protein selected from the group consisting of CD22, CD19, CD20, and CD21, and b) an immunogenic peptide comprising at least one T-cell epitope for use in vaccination of a subject against B-cell hyperproliferation or for use in the modulation of the immune response in a subject. The present invention further relates to a polynucleotide and a vector encoding said polypeptide and a host cell comprising the same. It also relates to a method for the stimulation of antigen-specific T-cells, comprising a) contacting antigen presenting cells (APC) with a polypeptide, the polynucleotide, or the vector of the invention, b) contacting said APC with T-cells, and c) thereby stimulating antigen-specific T-cells specific for said at least one T-cell epitope; to a method for immunizing a subject against B-cell hyperproliferation, to a method for immunizing a subject against an infectious agent, and to a method for inducing tolerance in a subject.

Claims

1. A method for stimulating antigen-specific T-cells to kill antigen presenting cells (APC) in a subject having an immune response to a T-cell epitope, comprising: (a) administering to the subject a polypeptide comprising: (i) a binding peptide that binds to at least one protein selected from the group consisting of CD22, CD 19, CD20, and CD21; and (ii) an immunogenic peptide comprising the T-cell epitope; (b) contacting the polypeptide with an APC of the subject; and (c) contacting the APC with T-cells, thereby stimulating the T-cells to kill the APC; wherein the immunogenic peptide comprises at least one T-cell epitope from a tumor antigen, and/or at least one T-cell epitope from a protein of a virus commonly infecting the subject or against which the subject has been vaccinated.

2. The method of claim 1, wherein the binding peptide is an antibody.

3. The method of claim 1, wherein the immunogenic peptide comprises at least one T-cell epitope from a latent gene of Epstein-Barr Virus (EBV).

4. The method of claim 1, wherein the modulation of the immune response is an activation.

5. The method of claim 1, wherein the binding peptide is an antibody directed to CD21 or a peptide comprising the CD21-binding peptide of EBV gp350.

6. The method of claim 1, wherein the modulation of the immune response is a repression.

7. The method of claim 1, wherein the binding peptide is a single-chain antibody.

8. The method of claim 1, wherein at least a part of the binding peptide is contiguous in amino acid sequence with the immunogenic peptide or with an adapter binding the immunogenic peptide.

9. The method of claim 1, wherein the APC are B-cells.

10. The method of claim 1, wherein the APC are lymphoblastoid cell lines (LCL).

11. The method of claim 1, wherein the T-cell epitope is a T-cell epitope for which the probability that T-cells present in a subject recognize the T-cell epitope is high.

12. The method of claim 1, wherein the T-cell epitope is from a protein of a virus commonly infecting a subject or against which a subject has been vaccinated.

13. The method of claim 1, wherein the stimulating of step (c) further comprises generating activated T-cells from the T-cells and killing the APC by the activated T-cells.

14. A method of treating B-cell hyperproliferation in a subject suffering therefrom, comprising: (a) contacting the subject with a polypeptide comprising: (i) a binding peptide that binds to at least one protein selected from the group consisting of CD22, CD19, CD20, and CD21; and (ii) an immunogenic peptide comprising at least one T-cell epitope, thereby treating said B-cell hyperproliferation in said subject; wherein the immunogenic peptide comprises at least one T-cell epitope from a tumor antigen, and/or at least one T-cell epitope from a protein of virus commonly infecting the subject or against which the subject has been vaccinated.

15. The method of claim 14, wherein the T-cell epitope is a T-cell epitope for which the probability that T-cells present in a subject recognize the T-cell epitope is high.

16. The method of claim 14, wherein the T-cell epitope is from a protein of a virus commonly infecting a subject or against which a subject has been vaccinated.

17. The method of claim 14, wherein the B-cell hyperproliferation is an EBV-associated disease.

18. The method of claim 17, wherein the EBV-associated disease is infectious mononucleosis, post-transplant lymphoproliferative disorder (PTLD), or B-cell lymphoma.

Description

FIGURE LEGENDS

(1) FIG. 1: T cell assay performed with EBV-transformed B cells (LCLs) used as antigen-presenting cells, pulsed with various amounts of antibodies fused with the 5H11 epitope from the EBV EBNA3C protein or with their respective heavy chain (HC). Antibodies tested are specific for CD21, CD19 and CD22. Positive controls include cells incubated with 5H11 peptide epitope, negative controls include anti-CD19, anti-CD21 or anti-CD22 antibodies devoid of antigens. Results are given in picograms IFN-gamma per ml.

(2) FIG. 2: T cell assay performed either with EBV-transformed B cells (LCLs) or the Burkitt's lymphoma cell line AG876 used as antigen-presenting cells, and pulsed with various amounts of CD21-specific antibodies fused with the 3H10 epitope from the EBV EBNA3C protein. Positive controls include cells incubated with 3H10 peptide epitope, negative controls anti-CD21 antibodies devoid of 3H10 or 3H10 anti-CD21 fusion proteins devoid of light chain. Results are given in picograms IFN-gamma per ml.

(3) FIG. 3: Treatment of EBV-transformed B cells (LCLs) and Burkitt's Lymphoma cell lines with polypeptides according to the invention comprising EBNA3C-3H10 leads to antigen presentation and efficient T cell activation. B cells were treated for 24h with 1 ng B-cell targeted antibodies (CD-21, -20, -19, and -22) loaded with EBNA3C epitope. Positive controls included cells pulsed with increasing amounts (1 ng-1 g) of 3H10 peptide alone, and negative controls included either untreated cells, or cells pulsed with antibodies not containing the EBNA3C-3H10 epitope. Following treatment, B cells were mixed with EBN3C-3H10-specific T cell clones at a ratio of 1:2. After 24h, secretion of IFN was measured by ELISA as an indicator of T cell activation. Results are given in pg/ml.

(4) FIG. 4: Antigen presentation by polypeptides according to the invention comprising EBNA3C-3H10 results in peptide-specific cell killing by CD4+ T cells. LCLs were treated with EBNA3C-3H10-loaded CD19 antibody (CD19-3H10 Ab; 1 ng and 10 ng), CD19 antibody containing no epitope (CD19 Ab), or EBNA3C peptide, or were left untreated. These target cells were then labeled with .sup.51Cr and co-incubated for 4h with EBNA3C-3H10-specific effector T cells at an increasing effector:target ratio (1:1, 3:1, 6:1, 12:1, 25:1, 50:1). A .sup.51Cr-release assay was performed to determine the % lysis of the target LCL population as a measure of specific cell killing.

(5) FIG. 5: General schedule of MCMV infection, lymphoma cell delivery and antibody treatment for mouse experiments of Example 5.

(6) The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

(7) Antibodies against CD21, CD19 and CD22 were fused with an antigenic epitope (5H11) from the Epstein-Barr virus latent antigen EBNA3C. The fusion proteins were used to introduce the epitope into the endosome of B cells transformed by the Epstein-Barr virus (lymphoblastoid cell lines, LCLs). The pulsed B cells were then co-cultured with a T cell clone that is specific for the EBNA3C 5H11 epitope. T cell activation was assessed by measuring interferon- release in the supernatant.

(8) LCLs directly incubated with the 5H11 epitope were used as positive controls. Antibody-5H11 fusion proteins devoid of light chains were used as negative controls, as were antibodies not fused with the epitopes. 1 ng of CD21-5H11 fusion proteins which carry 20 pg of 5H11 elicited an immune response comparable to the one obtained with 1 g of peptide (FIG. 1). Therefore, the efficiency of antigen presentation on activated B cells was 50.000 times higher after fusion with CD21 antibody than with the epitope alone. Similar results were obtained with the CD19, CD20, and CD22-specific antibodies. It is important to note that untreated EBV immortalized B cells cannot present 5H11 on the class II pathway.

EXAMPLE 2

(9) LCLs and AG876, a Burkitt's lymphoma cell line, were used as antigen-presenting B cells and their respective abilities to present the 3H10 epitope from the EBNA3C protein fused to CD21-specific antibodies was determined by an interferon- release assay. 3H10 peptides alone provided a positive control, non-functional fusion proteins devoid of heavy or light chains, mock-treated antigen-presenting cells were taken as negative controls.

(10) Both LCLs and AG876 presented 3H10 epitopes with a similar efficiency (FIG. 2). The AG876 presented 3H10 after incubation with the CD21 antibody-3H10 fusion protein less efficiently than LCLs but remained approximately 100 times more efficient than unconjugated 3H10. This relative decreased efficiency is consistent with defective antigen processing machinery in Burkitt's lymphoma cells. Nevertheless, the CD21 antibody-3H10 fusion protein elicited a potent immune response against the tumor cells.

EXAMPLE 2.1: FURTHER EVALUATING POLYPEPTIDES ACCORDING TO THE INVENTION IN EBV-TRANSFORMED B CELLS AND VARIOUS BURKITT'S LYMPHOMA CELLS LINES

(11) A panel of antibodies loaded with various EBV epitopes has been generated. These have been evaluated for their ability to present antigen to peptide-specific T cells and to activate these T cells. In FIG. 3 is shown one example of treatment with a polypeptide according to the invention comprising an epitope from the EBNA3C protein. We were able to show that treatment with these polypeptides according to the invention results in specific T cell activation in LCLs and in several Burkitt's lymphoma cell lines.

EXAMPLE 2.2 DETERMINING THE POTENTIAL FOR T CELLS ACTIVATED BY TREATMENT WITH POLYPEPTIDES ACCORDING TO THE INVENTION TO KILL THEIR TARGET CELLS

(12) Since polypeptides according to the invention can efficiently activate peptide-specific T cells, we wanted to determine whether these activated T cells are able to specifically kill the B cells presenting the epitopes from the immunogenic peptides. Here we performed .sup.51Cr-release assays to demonstrate that the activated T cells can indeed kill their targets. FIG. 4 shows one example of this in LCLs.

EXAMPLE 2.3: IN VIVO STUDIES IN A MOUSE LYMPHOMA MODEL

(13) A panel of polypeptides according to the invention in the form of armed antibodies containing T cell epitopes from common mouse pathogens are generated. B cell surface receptors CD-19, -20, -21 and -22, are targeted and the antibodies are coupled to the pp89 peptide, an immunodominant T cell epitope from the IE1 protein of mouse cytomegalovirus (MCMV). These antibodies are studied in the A20 model of mouse lymphoma. Injection of the A20 cell line into MCMV-positive BALB/c mice results in the development of disease that resembles human diffuse large B cell lymphoma (DLBCL).

(14) The serostatus of the animals to MCMV is assessed by serology prior to the commencement of the study. Seronegative animals are infected with MCMV in order to ensure seroconversion and priming of T cells against the MCMV pp89 peptide. All animals are re-infected with the virus 4 weeks prior to i.v. challenge with A20 lymphoma cells (FIG. 5). At days 5 and 15 following lymphoma cell delivery, animals are treated with the recombinant AgAbs containing pp89 peptides. Mice are subsequently monitored for survival and tumour development for 120 days following lymphoma cell challenge. Mice are sacrificed when external signs of suffering are present (such as reduced mobility and altered behaviour), as per the guidelines recommended by the Society of Laboratory Animal Science (GV-SOLAS), or if no adverse symptoms appear, at 120 days following injection of cells. Refer to FIG. 5 for a schematic representation of this experimental schedule.

(15) Molecular resonance imaging (MRI) is used in order to monitor tumour development at two time-points during the course of the study. Mill allows to both visualize the tumours and to perform volumetric analysis of the tumours. Imaging of all mice is performed when signs of tumour development are evident in the untreated group, and again prior to sacrifice. In addition, anatomical and histological examinations are performed upon sacrifice of the mice.

(16) Using the experimental schedule outlined in FIG. 5, a panel of antibodies and treatment regimes is investigated. Firstly, antibodies against the full panel of B cell surface receptors, CD19, -20, -21 and -22 are tested, using antibody and adjuvant (Poly I:C) co-treatment. Peptide-loaded and unloaded antibodies are compared in targeting these surface receptors. Tumour growth and animal survival are monitored as markers of treatment efficacy, and are compared relative to untreated control animals and animals without lymphoma. Further experiments include: i) determination of the most effective dose of antibody treatment; and ii) an evaluation of the efficacy of treatment with armed antibodies and CD20/rituximab co-injection. Indeed, antibodies directed against CD21 or CD19 have been found to evince low cytotoxic properties that could be instead provided by anti-CD20 antibodies and therefore combine two different angles of attack against the lymphoma cells.

(17) Once a panel of antibodies has been tested in the A20 lymphoma model, studies are extended to other lymphoma models. This includes the BCL1 model in which BCL1 cells can induce a DLBCL-like or CLL-like lymphoma, depending on the route of inoculation (i.p. or i.v, respectively), and a Burkitt's lymphoma-like model using cells from B6-myc transgenic mice.

EXAMPLE 3

(18) Presentation of microbial antigens at the surface of B lymphocyte cells elicits recognition and destruction through T cells specific to these antigens. These T cells are present in most individuals who were previously infected by common viruses, such as herpesviruses. Individuals with a chronic Hepatitis C or HIV infection carry an increased proportion of activated B cells that can efficiently present antigens (Moir and Fauci, 2009, Nat Rev Immunol; Sugalski, Rodriguez, Moir, Anthony, 2010, J. Immunology). These infectious agents have an intrinsic ability to modify their surface antigens and subsequently they are able to evolve faster than the host's immune system can adapt. As a result, infected patients cannot clear their infections.

(19) To overcome this problem, a library of polypeptides is generated, comprising anti-CD21 antibodies coupled to a library of Hepatitis C antigens that are found in patients with a long-standing infection, which covers the spectrum of viral antigens that appear in the course of infection and include all stages of virus evolution. This antibody library is administered to patients with a recently acquired Hepatitis C and thus primes the patient's immune system against all possible virus variants and therefore enables their elimination.

(20) The same method is applied to patients recently infected with HIV, using a library of HIV antigens that are found in patients with a long-standing infection, which covers the spectrum of viral antigens that appear in the course of infection and include all stages of virus evolution.

EXAMPLE 4

(21) An antibody fusion protein is created, comprising an anti-CD21 antibody fused to an immunodominant peptide from a common viral or bacterial pathogen, for example EBNA3C from EBV, and produced according to conventional methods. The antibody fusion proteins are administered to patients suffering from B-cell lymphoma, where they are taken up by Lymphoma cells. The Lymphoma cells present the T-cell epitopes comprised in the EBNA3C peptide and thus activate EBNA3C-specific T-cells, which in turn eliminate the presenting Lymphoma cells. CD4+ T cells can also act as cytotoxic T cells to orchestrate the killing of target cells. There is also the possibility of cross-presentation to CD8+ T cells.

EXAMPLE 5

(22) An antibody fusion protein is created, comprising an anti-CD22 antibody fused to myelin basic protein (MBP). The fusion protein is applied to patients at a high dose. Thus, tolerance to MBP is induced and thus progression of Multiple Sclerosis is reduced.