The present invention relates to an adjuvant derived from human lymphocytes. The adjuvant can be used in combination with traditional vaccines or cancer immunotherapy, to enhance the response of the patient's immune system to the vaccine or other immunotherapeutic agent. The adjuvant is derived from the supernatant collected from cultured activated lymphocytes.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to an adjuvant derived from human lymphocytes. The adjuvant can be used in combination with traditional vaccines or cancer immunotherapy, to enhance the response of the patient's immune system to the vaccine or other immunotherapeutic agent.

BACKGROUND INFORMATION

Immunological adjuvants are used in combination with vaccines to augment the immune response to the antigen. One way in which immunological adjuvants function is by attracting macrophages to the antigen, so that the macrophages can present the antigen to the regional lymph nodes and initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the antigen, or may influence the immune response by other mechanisms such as depot effect, cytokine induction, complement activation, recruiting of different cell populations of the immunological system, antigen delivery to different antigen presenting cells, regulation of the expression of HLA class I or class II molecules and the stimulation to produce different antibody subtypes. Many of the newer vaccines are only weakly immunogenic and thus require the presence of adjuvants.

Materials having adjuvant activity are well known. Alum (Al(OH).sub.3), and similar aluminum gels are adjuvants licensed for human use. The adjuvant activity of alum was first discovered in 1926 by Glenny (Chemistry and Industry, Jun. 15, 1926; J. Path. Bacteriol, 34, 267). Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.

Other materials are also known to have adjuvant activity, and these include: Freund's complete adjuvant, a water-in-mineral-oil emulsion which contains killed, dried mycobacteria in the oil phase; Freund's incomplete adjuvant, a weaker formulation without the mycobacteria; saponin, a membrane active glucoside extracted from the tree Quillia saponaria; nonionic block copolymer surfactants, non-metabolised synthetic molecules which tend to bind proteins to cell surfaces; ISCOMS, lipid micelles incorporating Quil A (saponin) which mimic, in physical terms, infectious particles; and muramyl dipeptide, a leukocyte stimulatory molecule that is one of the active components of killed mycobacteria. A known adjuvant in cancer therapy is bacillus calmette guerin (BCG) which is used in combination with various anti-cancer vaccine strategies. GM-CSF has also been found to be an effective adjuvant when used in combination with autologous tumor cells.

With all of these agents, toxicity, unacceptable chronic reactions and/or low potency (in the case of BCG) are features which currently limit their use as potential adjuvants. Thus there is an ongoing and current need for new adjuvants to boost the human immune response to vaccines, in both cancer therapy and other disease treatments.

One line of research in the development of adjuvents has been directed to the study of dendritic cells. Dendritic cells (DC) are professional antigen presenting cells (APC) that have the unique capacity to initiate primary immune responses in vivo and in vitro (1-3). They are derived from myeloid (DC1) or lymphoid (DC2) precursors and are distributed in their immature form throughout the body in tissues that commonly encounter environmental pathogens (skin, mucus membranes, gut epithelia, etc.) (1,2,4-7). Whereas DC1 and DC2 comprise a small percentage of the total number of mononuclear cells in the peripheral circulation, DC1 precursors in the form of CD14+/CD11c+/HLA-DR+ monocytes are relatively abundant, constituting about 10% to 15% of mononuclear blood cells (11-15).

Immature DC express a host of surface structures that are involved in antigen acquisition, DC activation/maturation, and antigen presentation (1,2,8). Once DC encounter antigen, they undergo a maturation process characterized by the up-regulation of HLA class I and II molecules as well as co-stimulatory molecules and interact with cognate receptors on T and B lymphocytes, resulting in the generation of antigen specific cellular and humoral immune responses (1,2,9,10).

DC are considered to be the primary APC in the immune system. The ability to isolate these cells and/or their precursors and to study them in vitro has added considerable dimension to knowledge of their role in innate and acquired immunity (1,2). The classic means of generating human DC in vitro is to isolate and enrich CD14+-monocytes from peripheral blood and culture them for various periods of time in GM-CSF and IL-4 followed by final maturation with a number of cytokines, including IL-2, IL-6, IL-7, IL-13, IL-15, TNF.A-inverted., IL-1.E-backward., (16,36) or with various other agents including lipopolysaccharides, PGE.sub.2, type 1 interferons, or double-stranded RNA (20-24).

Numerous investigators have shown that these in vitro generated monocyte-derived DC are potent antigen presenting cells (APC) capable of initiating primary and recall antigen-specific CD4+ and CD8+ T cell responses (27-30). Recent in vitro studies have generated a rather extensive body of information regarding the biology of DC1 and shed light on the processes whereby antigen specific immune responses are generated in vivo (1-2). In the peripheral tissues, immature DC acquire antigenic materials in the context of danger signals initiating a complex cytokine/chemokine milieu that is generated by DC and other cell types in the vicinity (31). Soluble mediators produced by DC may act in an autocrine or paracrine fashion. T cells produce additional cytokines and chemokines following interaction with antigen armed DC, as do other immune cells that are activated by the cytokines released (32-35). This complex network of interactions may in turn create an environment that promotes the generation of DC from their monocyte precursors.

Several investigators have described the use of various cell-free culture supernatants, also referred to as "conditioned media" as DC maturation agents. These media contain more or less well defined mixtures of cytokines (12,25,26). Monocyte conditioned media (MCM), containing IFN.A-inverted., IL-1.E-backward., IL-6, and TNF.A-inverted., has been shown to induce expression of CD83 and p55, surface molecules that are characteristic of mature DC (26). However, when combinations of these cytokines were added to immature DC at concentrations comparable to those found in the conditioned media, they were less effective in maturing DC compared to MCM. These results suggest that additional components were required to affect full maturation of immature DC.

In one study, Kato et al prepared conditioned media (designated TCCM) by culturing isolated T cells with anti-CD3 monoclonal antibodies that had been adhered to plastic surfaces (25). This media was able to mature immature DC that had been generated from monocytes cultured in (GM-CSF and IL-4. Interestingly, different clones of anti-CD3 induced different quantities of soluble CD40 ligand and IFN (and these differences were reflected in the capacity of the media to mature DC.

Whereas MCM and TCCM are very effective mediators of final DC maturation, their capacity to differentiate monocytes into immature DC was not reported. The inventors are aware of one report where this activity was observed with media from PBMC stimulated with CpG-A oligonucleotides (33). It is well established that CpG-A induces type 1 interferons (IFN.A-inverted./IFN.E-backward.) production by plasmacytoid DCs, a minor cellular component in PBMC (6, 37-39). In the cited study, antibodies to IFN.A-inverted. diminished but did not abrogate the activity of this culture media suggesting that additional cytokines might be participating in the induction of monocyte differentiation. This is certainly possible since IFN.A-inverted. is known to induce production of cytokines in other cell types (including T cells) that may affect monocyte differentiation (6,38,39).

It is thought that compounds or compositions which promote that maturation of dendritic cells, when administered in combination with a vaccine antigen, will result in more antigen presenting cells presenting the vaccine antigen to T lymphocytes and B cells, thus bolstering the immune response to the vaccine antigen.

SUMMARY OF THE INVENTION

The present invention solves the above need by providing new adjuvants, based on products of human lymphocytes, that provide immunological potentiation and increase the amount and quality of the immune response to vaccine antigens.

In one aspect of the invention, the adjuvant is derived from supernatant material collected from in-vitro stimulated cultured human peripheral blood mononuclear cells. Naive T-cells are activated during the culture process. The adjuvant works to enhance the ability of a vaccine to initiate, create, boost and/or sustain an immune response to an agent in humans, and other animal or plant species.

The present invention provides an adjuvant based on a mixture of cytokines and chemokines obtained from peripheral blood mononuclear cells stimulated with antiCD3/CD28-coated beads. As used herein, the term "lymphocyte conditioned medium (LCM)", will be used to refer to this adjuvant. It has been found that LCM is a highly effective conditioned medium with the capacity to mature monocyte-derived DC and to render monocytes into potent APC. The adjuvant can provide a rapid, cost-effective, and probably more "physiologic" method to derive large amounts of DC1 from precursor cells in vitro, and LCM can therefore function as an effective vaccine adjuvant. It is thought that PBMC-derived products may provide the cytokine milieu required to rapidly generate DC1 from their precursors after the initiation of an immune response in vivo.

The cytokines and chemokines identified in the LCM preparations are known to participate in the generation of immune responses by their autocrine or paracrine effect on APC and responding T and B cells. The concentrations of cytokines found in the LCM are considerably lower than the concentrations of cytokines that are commonly used to differentiate monocytes into immature DC or to mature DC in vitro (16,22). LCM contains cytokines (IFN, IL-12) and soluble CD40 ligand that are known to polarize T cells towards a TH1 response as well as cytokines (IL-4 and IL-10) that polarize T cells towards the TH2 responses (5,40,41). These latter cytokines may also induce T cell anergy when present in cultures of antigen presenting immature DC and T cells. However, the presence of IL-10 and the small amounts of IL-4 in the LCM did not abrogate a T cell recall response to TT; rather, the T cell responses were augmented, clearly demonstrating that the effect of TH1 cytokines dominates.

In addition to proinflammatory cytokines, high concentrations of chemokines were detected in LCM. These chemokines are produced by lymphoid cells as well as by non-lymphoid cells in the context of an inflammatory process (35,42). As an example, RANTES is produced by CD8+-T cells and it in turn induces the generation of other cytokines and chemokines (MIP1.E-backward., IL-2, IL-6, and type 1 interferons) that activate T cells as well as monocytes (43). The induction of these cytokines and chemokines might be representative of what occurs when T cells are activated by APC in vivo in the context of antigen presentation. Following T cell activation through T cell receptor and CD28 ligation, the T cells release cytokines and chemokines that are known to influence the differentiation of monocytes into immature DC as well as their migration to regional lymphoid organs. These soluble factors may also attract DC precursors and other APC to the environment of initial antigen encounter (danger signal). Together the cytokines and chemokines produced by activated T cells and, downstream, by bystander cells could be expected to magnify the immune response cascade, a desirable property of an adjuvant. There is an increasing awareness of the capacity of various cytokines to act as adjuvants for vaccines, in particular GM-CSF and IL-2.

The generation of a PBMC-derived conditioned medium has the capacity to generate large amounts of immature DC from their precursors and to mature monocyte-derived DCs. This rapid and cost-effective method can play an important role in the development of future vaccines, and for use as an adjuvant. In addition, the wide range of cytokines and chemokines contained in LCM suggests a more physiologic stimulus, providing a cytokine milieu similar to what might be found in vivo once T cells encounter antigen.

The present invention provides a method of using the adjuvant, both supernatant and cell-based, in combination with a vaccine antigen, to provide an enhanced immune response to the vaccine.

It is an object of the present invention, therefore, to provide a vaccine adjuvant capable of enhancing immunogenic response to the vaccine.

It is a further object of the invention to provide a vaccine adjuvant derived from human lymphocytes.

It is a further object of the invention to provide a vaccine adjuvant derived from supernatant collected from stimulated cultured human lymphocytes.

It is an additional object of the invention to provide a method of using the vaccine adjuvant, by administering the adjuvant to a host animal in combination with a vaccine.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect, the present invention provides a method of enhancing the immune response to a vaccine antigen in a host mammal, comprising administering lymphocyte conditioned medium, the supernatant derived from activated human lymphocyte cells cultured with growth media, in combination with the vaccine antigen. Preferably, the mammal is a human. Culture methods and protocol are standard and known in the art. Human (or other mammal, depending on the mammal to be treated) peripheral blood mononuclear cells (PBMC) obtainable from any source are diluted in commercially available tissue culture growth media. The cells are incubated with an activation agent consisting of beads coated with antibodies to CD3/CD28. On about day 3, cells and beads are separated from the culture media and the cells and beads are resuspended in additional growth media as needed. To harvest the cells, they are resuspended in centrifuge tubes and pelleted, after which the supernatant can be drawn off with a pipet and stored for later use.

As used herein, the term "supernatant" refers to the liquid drawn off the cultured cells, in the manner described above. "Lymphocyte conditioned medium" and "supernatant" are used herein interchangeably, and refer to the liquid drawn off the cultured cells. Studies have been carried out to characterize the supernatant, and it has been found to contain molecules having an average molecular weights of less than about 100,000 daltons.

Administration is by known methods used for vaccination, and suitable delivery methods include, but are not limited to, intramuscular, intercutaneous and subcutaneous injection. Typically, about 10 .mu.g to about 500 .mu.g are administered in combination with the antigen. Administration can be weekly, biweekly, monthly or yearly, depending on the antigen and the level of immune response desired. Enhancement of the immune response can be observed, for example, by conducting standard assays known in the art that assess cellular immunity (such as T cell proliferation) and measure antibody titres post immunization.

The present invention is suitable for use with a large variety of vaccines, including, but not limited to, measles, mumps, rubella, influenza, haemophilus influenzae type B vaccines, diphtheria, tetanus, pertussis, pneumococcal polysaccharide vaccines, meningococcal polysaccharide vaccines, staphylococcus aureus vaccines, respiratory syncytial virus, streptococcus, parainfluenza mycoplasma pneumoniae, mycobacterium leprae, nocardia, legionella, pseudomonas, cholera vaccines, typhoid fever, poliovirus, hepatitis A vaccine, rotavirus, escherichia coli, shigella, hepatitis E, listeria, giardia lamblia, toxocariasis, trichuriasis, ascariasis, amebiasis, cysticercosis, hepatitis b recombinant and plasma-derived vaccines, HIV-1 and HIV-2; HTLVI and HTLV-II, Epstein-Barr, hepatitis C, herpes B, human papillomavirus, herpes simplex type 1 and 2, chlamydia, gonorrhea, treponema (syphilis), anthrax, rabies, schistosomiasis, plague, yellow fever vaccines, japanese encephalitis and tick-borne encephalitis vaccines, malaria, leishmaniasis, lyme disease, lymphatic filariasis and onchocerciasis, trypanosomiasis and chagas' disease, rickettsia and typhus fevers, dengue fever, adenovirus vaccines, varicella zoster vaccines, cytomegalovirus, coronaviruses and rhinioviruses, streptobacillus, allergy peptide, infectious disease peptide vaccine, cancer peptide vaccine, autoimmune peptide vaccine, and cancer vaccines utilizing antigen, peptide, DNA fragments and/or any other molecular species on the surface or within the cancer cell.

Claim 1 of 12 Claims

1. A composition for enhancing an immune response to an antigen in a mammal, comprising an antigen in combination with lymphocyte conditioned medium, said lymphocyte conditioned medium is the supernatant derived from cultures of peripheral blood mononuclear cells activated with anti-CD3- and anti-CD28-coated beads, wherein said composition when administered to a mammal facilitates and enhances dendritic cell maturation and antigen-presenting cell function which results in enhanced immunologic responses to said antigen in the mammal.
 

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