The applicants claimed to use LMP1 and LMP1 fusion proteins (LMP1-CD40 and others) as adjuvants in vaccination therapies against microbes and tumors, in the referred patent application. By adjuvant, they refer to an immunostimulant that triggers activation of antigen-presenting cells (APC), such as dendritic cells (DC), macrophages and B cells. Though the adjuvant effect of LMP1 fusion proteins can be claimed as their invention, LMP1 native protein had already been demonstrated to have adjuvant function by others, much earlier than their application filing date (Rowe, Eur J Immunol 1995; Smith, Blood 2009). Specifically, these prior studies showed that LMP1 expression in B cells and epithelial cancer cells makes them present experimentally transduced antigens and be killed by T cells. Throughout this patent application (and their claims), there is no evidence (data) that the applicants identified any novel function of LMP1 beyond what had been known in the prior literature. Is such an "obvious" function of LMP1 patentable as claimed in the application?
As one of the inventors, I thank the questioner for taking notice of our patent application and thereby implicitly recognizing the importance of the invention. As was known, infection with Epstein-Barr virus (EBV) can result in acute infectious mononucleosis (“AIM” or “Mono”). In this condition, the body mounts the strongest CD8+ T cell response ever described for humans. By tetramer analysis, up to 44% of the CD8+ T cells in the blood recognize a single peptide epitope (RAKFKQL) from an EBV lytic phase protein (BZLF1) (Callan, M.F., et al., J Exp Med, 1998). Thereafter, EBV infection enters a chronic phase in healthy individuals in whom up to 5.5% of CD8+ T cells in the blood recognize this single peptide epitope for life (Tan, L.C., et al., J Immunol, 1999). Given the importance of CD8+ T cells for cancer immunotherapy and the defense against viruses (e.g., influenza, ebola, etc.) and microbes (e.g., malaria), we tried to learn from EBV in order to make much needed vaccines and therapies.
Prior to the invention, we had published many papers on the role of CD40 as an activator of immune cells (dendritic cells (DCs), macrophages, and B cells) and had designed novel (now patented) forms of soluble CD40 ligand (CD40L) that can be used as vaccine adjuvants and cancer immunotherapy agents. In parallel, other scientists reported that stimulation of the CD40 receptor on DCs is essential for CD8+ T cell responses. Consequently, we were intrigued by reports that the latent membrane protein-1 (LMP1) of EBV functioned like a constitutively active form of the CD40 receptor. As described in our patent application and our post-filing publications, the LMP1 nucleic acid sequence can be extracted from EBV and used in a variety of vaccine formats as a “portable genetic adjuvant.”
In the specification, we highlighted novel aspects of the invention:
 However, prior to the present invention, it was not recognized that LMP1 or LMP1-CD40 could be used as adjuvants and there was no suggestion as to how this might be accomplished. One of the central, non-obvious, and inventive aspects of the present invention is that it envisions LMP1 or LMP1-CD40 as portable adjuvant compositions that can be introduced into microbes, most preferably viruses, or tumor cells. These microbes or tumor cells carry their own antigens with them. By placing LMP1 or LMP1-CD40 directly into them, the antigen and adjuvant become co-extensive in space and time, which is an important feature of the present invention.
However, the questioner points to two publications that are alleged to make our invention obvious and unpatentable:
(1) Rowe, Eur J Immunol 1995. This group used a CD8+ T cell clone from an EBV seropositive donor (NB36) to recognize target cells expressing a peptide epitope from the EBNA3A protein (YPLHEQHGM) in the context of HLA-B35. To study CD8+ T cell recognition of latently infected Burkitt lymphoma (BL) cell lines and other B cell lines, they transduced these cells with a vaccinia virus expressing EBNA3. The results were striking. The BL cell lines were essentially invisible to the immune system because they had a low level of peptide transporter-associated proteins (Tap-1 and Tap-2) and a low level of HLA Class I synthesis. Thus, they did not express peptide/MHC-I on their surface. However, if LMP1 was introduced into these cells, now the BL cells did express peptide/MHC-I on their surface and could be recognized and killed by the NB36 CD8+ T cells. Hence the title of their paper, “Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression.”
(2) Smith, Blood 2009. This group reported similar findings to Rowe et al. using different EBV-specific CD8+ T cell lines isolated from EBV seropositive subjects. They used a cervical carcinoma epithelial cell line (C33A) to show that transfection with a plasmid for LMP1 upregulated the expression of cell surface MHC-I. Even so, the LMP1-transfected C33A cells were poorly recognized by CD8+ T cells specific for a peptide epitope from LMP1 (YLQQNWWTL) that had been generated from an EBV-infected donor (i.e., poor antigen presentation in “cis”). A different result was obtained when the C33A cells were transfected with two plasmids: one for LMP1 as before and the other expressing LMP2 (a different EBV protein). In this case, LMP2-specific CD8+ T cells recognizing a peptide epitope from LMP2 (CLGGLLTMV) showed much better recognition of C33A cells transfected with LMP2 when LMP1 was also present (i.e., LMP1 augments antigen presentation in “trans”). In this case, self-aggregation by the N-terminus of LMP1 was required. Hence the title of their paper, “Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation.”
We disagree that our invention is obvious. Obviousness is determined in relationship to the invention as claimed and as understood by a person having ordinary skill in the art (PHOSITA). With this in mind, consider the two independent claims in US20130039942 A1 being examined at USPTO:
“8. (Currently Amended) An isolated virus, microbe, or host cell, comprising:
(i) a first expression cassette for expressing a protein, comprising a multimerizing domain operatively joined to a cytoplasmic signaling domain of a receptor such that the protein assembles into a complex of three or more protein moieties, and, wherein when the protein is LMP1, then the nucleic acid encoding LMP1 does not contain introns; and
(ii) a second expression cassette for expressing an antigen, with the proviso that the protein is not an Epstein-Barr virus (EBV) protein.”
For claim 8, neither Rowe nor Good describe an antigen that does not come from EBV. These references do not anticipate the claim limitation to non-EBV antigens. As explained in MPEP 2131, “To anticipate a claim, the disclosure must teach every element of the claim.” Thus, claim 8 remains standing as novel.
- (Currently Amended) An immunogenic composition, comprising: a delivery system comprising a microorganism other than Epstein Barr virus (EBV);
an antigen comprising a tumor antigen, viral antigen, or microbial antigen, wherein the antigen is endogenous to the microorganism used to deliver the immunogenic composition or not normally expressed by the microorganism used to deliver the immunogenic composition, and wherein the antigen is not an EBV protein; and an adjuvant comprising the protein of claim 8 (i).
Of note, claim 16 is directed toward an “immunogenic composition,” a term that is defined in the specification as: “ The term "immunogenic composition" or "immunogen" refers to a substance that is capable of provoking an immune response.”
For claim 16 drawn to an immunogenic composition, neither Rowe nor Good used a system that “provoked” an immune response. Instead, they used CD8+ T cell lines generated from subjects who had already been infected with EBV. These CD8+ T cell lines were simply used as indicator cells to determine if peptide/MHC-I complexes were present on the surface of a cell in vitro. In contrast, our patent application showed how LMP1 was able to activate primary human DCs such that they induced CD8+ T cell responses against HIV-1 Gag antigen, a non-EBV antigen. This reduction-to-practice is shown in Fig. 15 which depicts the experimental protocol and shows the data from these in vitro immunization experiments. Of note, the immune cells (PBMCs) were taken from HIV seronegative subjects so that the anti-HIV CD8+ T cell responses were truly de novo and not merely a “re-stimulation” of existing antigen-specific CD8+ T cells as performed by Rowe and by Good. As an in vivo example, Fig. 18 showed that DNA vaccination with a plasmid for LMP1 plus a plasmid for the gp100 tumor antigen was able to protect mice from lung metastases by B16F10 melanoma, a highly aggressive and difficult to treat tumor.
Since Rowe and Good simply used CD8+ T cells as indicator cells, they did not show that LMP1 enhanced immunogenicity, which is a much more meaningful property than “antigen processing” (used in the title of Rowe) or “antigen presentation” (used in the title of Good). In particular, antigen presentation does not always generate immunity but can also generate tolerance, as shown by Bonifaz, L. et al. (Ralph Steinman, senior author), “Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance,” J Exp Med 196:1627-1638, 2002). Thus, according to Ralph Steinman (Nobel Prize 2011), the presence of antigen processing or antigen presentation alone is not reason enough to say that an immunogenic composition has been achieved. By our definition of “immunogenic composition” at , an LMP1-containing immunogenic composition must “provoke” an immune response and neither Rowe nor Smith showed this.
We remain confident about the validity of our patent application. We are excited about using LMP1 and its derivatives for cancer immunotherapy and as vaccine adjuvants. In so doing, we will be applying one of the strongest immune stimulants ever described and one that is already present in the 90% of us who are both healthy and EBV seropositive.