WO1990003985A1

WO1990003985A1 - A differentiation antigen, nda5, associated with amplification of differentiation of t and b lymphocytes

Info

Publication number: WO1990003985A1
Application number: PCT/US1989/004134
Authority: WO
Inventors: Nicole Suciu-Foca; Donald W. King
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 1988-10-14
Filing date: 1989-09-22
Publication date: 1990-04-19
Also published as: AU4334389A

Abstract

This invention provides a purified, differentiation antigen designated NDA5 associated with the amplification of the proliferative responses of T and peripheral B lymphocytes to mitogens and antigens, characterized by being a heterodimer having an apparent molecular weight under non-reducing conditions of about 170-180 kd, by being comprised of two subunits having apparent molecular weights of about 75 kd and 85 kd, and by being immunologically reactive with the monoclonal antibody designated MoAb NDA5 (ATCC Accession No. HB 9848). This invention also provides a method of augmenting the responsiveness of T and peripheral B lymphocytes in response to a mitogen and an antigen which comprises contacting T and peripheral B lymphocytes under suitable conditions with an antibody directed against NDA5 in the presence of the mitogen or antigen so as to effect an augmented response by the T and peripheral B lymphocytes.

Description

A Differentiation Antigen, NDA5, Associated with Amplification of Differentiation of T and B Lymphocytes

The invention described herein was made in the course of work under Grant No. R01-AI25210-02 from the National Institute of Health. The U.S. Government has certain rights in this invention.

Background of the Invention

A number of pathalogical disorders in humans compromise the ability of the immune system to properly respond to antigenic stimuli, thus rendering an inflicted patient more susceptible to harmful infections. Such disorders include AIDS, chronic lymphocytic leukemia (CLL) , myelocytic leukemia and multiple myeloma.

Understanding the factors which regulate the interaction and proliferation of lymphocytes is of obvious importance in the understanding and eventual cure of such disorders.

The transition of T and B lymphocytes from their resting state to a state of functional maturity involves distinct steps of activation, proliferation and differentiation, each step requiring specific signals. Activation is initiated after antigens, mitogens, or antibodies interact with the T-cell antigen receptor complex or with B-cell surface immunoglobulins. Recently, non-lineage-specific leukoyte-common antigens (LCAs) have received increasing attention due to the evidence that such molecules can play an important role in conjugate formation and outer communication between cells of different lineage as well as the regulation of lymphocyte adhesion, all processes being of central importance to. proper immunological function.

The currently recognized LCAs include CD43 (gp 95) , CD44 (gp 66-85) , CD45 (1200) , CD18 and CDlla representing the alpha (A50 to 175 kd) and the beta (gp 95) chains of the lymphocyte function-associated antigen (LFA-1) .

Two new differentiation antigens (NDA3 and NDA4) have recently been described which are expressed by normal T and B lymphocytes following activation with mitogens or antigens, and which are also present on lymphoblastoid T and B cell lines transformed by HTLV-1 and Epstein- Barr Virus, respectively.

Monoclonal antibodies MoAb NDA3 and MoAb NDA4 recognize NDA3 and NDA4, respecitvely, and stimulate the growth and differentiation of activated human B cells. They thus appear to mimic the effect of lymphokines, possibly by reacting with cell surface receptors for such factors.

The subject invention discloses a new differentiation antigen, NDA5, which is a LCA involved in conjugate formation and lymphocyte interaction, and which differs in size from both NDA3 and NDA4.

The subject invention also discloses a monoclonal antibody MoAb NDA5 which is specific for NDA5 and which augments the responsiveness of both T or peripheral B lymphocytes to mitogens and antigens.

Summary of the Invention;

This invention provides a purified, differentiation antigen designated NDA5 associated with the amplification of the proliferative responses of T and peripheral B lymphocytes to mitogens and antigens, characterized by being a heterodimer having an apparent molecular weight under non-reducing conditions of about 170-180 kd, by being comprised of two subunits having apparent molecular weights of about 75 kd and 85 kd, and by being immunologically reactive with the monoclonal antibody designated MoAb NDA5.

This invention further provides an antibody directed to, and capable of specifically forming a complex with, the purified differentiation antigen designated NDA5.

This invention also provides a method of augmenting the responsiveness of T and peripheral B lymphocytes in response to a mitogen and an antigen which comprises contacting T and peripheral B lymphocytes under suitable conditions with an antibody directed against NDA5 in the presence of the mitogen or antigen so as to effect an augmented response by the T and pheripheral B lymphocytes.

This invention still further provides a method of potentiating an immune response to a specific antigen which comprises contacting T or peripheral B lymphocytes with a mixture comprising the specific antigen and an antibody directed against NDA5 in an amount effective to potentiate the immune response. Detailed Description of the Invention;

This invention provides a purified, differentiation antigen designated NDA5 associated with amplification of the proliferative responses of T and peripheral B lymphocytes to mitogens and antigens, characterized by being a heterodimer having an apparent molecular weight under non-reducing conditions of about 170-180 kd, by being comprised of two subunits having apparent molecular weights of about 75kd and 85kd, and by being immunologically reactive with the monoclonal antibody designated MoAb NDA5.

The hybridoma cell line, NDA5, was deposited pursuant to, and in satisfaction of the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession No. HB 9848.

The NDA5 is likely a non-lineage-specific, leukocyte- common antigen (LCA) . Preferably, the T and peripheral B lymphocytes are human lymphocytes.

The invention further provides an antibody directed to, and capable of forming a complex with, purified NDA5. This antibody may be a monoclonal antibody, or more specifically, MoAb NDA5. The method of preparation of MoAb NDA5 by immunization of a mouse with human T lymphocytes is discussed in detail in the Experiments.

Monoclonal antibodies directed against NDA5 may be obtained from a hybridoma which produces a monoclonal antibody directed against said antigen. In one embodiment of the invention, MoAb NDA5 is produced by a hybridoma designated NDA5 (ATCC Accession No. HB9848) .

A hybridoma cell producing a monoclonal antibody directed against NDA5 may be formed by fusing an NDA5 monoclonal antibody-producing cell and an immortalizing cell line, that is, a cell line that imparts long-term tissue culture stability on the hybrid cell. The anti- NDA5 monoclonal antibody-producing cell used in the fusion may be a spleen cell of an animal immunized against NDA5.

This invention further provides a method of augmenting the responsiveness of T and peripheral B lymphocytes to a mitogen and an antigen which comprises contacting T and peripheral B lymphocytes under suitable conditions with an antibody directed against NDA5 in the presence of the mitogen and antigen so as to effect an augmented response by the T and peripheral B lymphocytes. Preferably, the T and peripheral B lymphocytes are human lymphocytes.

T and peripheral B lymphocytes normally respond to stimulation by mitogens and antigens by either maturing, proliferating or both. NDA5 is capable of enhancing the effects of antigens and mitogens on T and peripheral B cells if the cells are stimulated with a mitogen and an antigen and then contacted with MoAb NDA5. Alternatively, T and peripheral B lymphocytes may be contacted with MoAB NDA5 prior to or at the same time as they are contacted to the antigen or mitogen. Time constraints as well as other parameters effecting the behavior of MoAb with T and peripheral B lymphocytes are discussed in the Experiments. In one embodiment of the invention, the T and peripheral B lymphocytes are from a patient whose immune system is compromised. In this case, it would be advantageous to augment the responsiveness of T and peripheral B cells to antigens and mitogens in order to circumvent the potentially devastating physiological effects of such disorders. A patient whose immune system is compromised could be infected with HIV, or have chronic lymphocytic leukemia, myologenous leukemia, or multiple myeloma.

There are a number of naturally occurring antigens as well as mitogens which are capable of stimulating an immune response. In one embodiment of this invention, an antigen may be a soluble antigen, such as a purified protein derivative of Microbacterium Tuberculosis (PPD) or Tetanus Toxoid, as an allogenic stimulator of primary and secondary mixed lymphocyte cultures. A mitogen may be a plant mitogen, such as pokeweed mitogen (PwM) .

Finally, this invention discloses a method of potentiating an immune response to a specific antigen which comprises contacting T or peripheral B lymphocytes with a mixture comprising the specific antigen and an antibody directed against NDA5 in an amount effective to potentiate the immune response. Preferably, the T and peripheral B lymphocytes are human lymphocytes.

In one embodiment of the invention, MoAb NDA5 can be used in conjunction with a specific antigen as an adjuvant in vivo. This may be effective at potentiating the generation of immune responses to specific antigens, such as a viral antigen (e.g. HTLV- III surface protein) .

Alternatively, MoAb NDA5 can be used in conjunction with a specific antigen as an adjuvant in vitro. An in vitro use of and adjuvant comprising MoAb NDA5 may be the in vitro treatment of a subject's bodily fluids or tissues with this adjuvant to eliminate an undesirable antigen.

Certain embodiments of this invention are exemplified in the Examples which follow. The Examples are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.

Example 1

Production of MoAb NDA5

MoAb NDA5, (IgM) was produced by immunizing mice with alloreactive human T cell clones, and fusing the splenocytes with the NS1 plas ocytoma, as previously described. The antibody was used as serial dilutions of culture supernatant. Other murine MoAbs used as controls were propagated and tested under identical conditions.

Example 2

Preparation of Cells

PBMC were obtained by Ficol-Histopaque gradient centrifugation of whole blood from healthy individuals or from patients. PBMC were first depleted of monocytes by plastic adhesion, and then applied to a nylon wool column. Adhering (B cells) and non-adhering cells (T cells) were recovered by intensively washing the column.

For further purification of T and B lymphocytes Lympho- Kwik-T and Lymphokwik-B were used as described by the manufacturer (One Lambda, Inc., Los Angeles, Ca.). Mitogen, soluble antigen and MLC-stimulation was induced as previously described. Cytofluorometric studies were performed on a FACS Star and Ortho- Cytofluorometer using direct and indirect i munofluorescence methods. Example 3

Immunofluorescence Studies of Cell Surface Distribution of NDA5

Immunofluorescence studies showed that NDA5 is present on peripheral blood ononuclear cells but is absent from platelets. Analysis of purified T cell, B cell, monocyte and granulocyte suspensions demonstrated the presence of this antigen on the membranes of >95% of the cells from each of these populations. The NDA5 antigen was found on all the cells from 5 different EBV-transformed B cell lines, Burkitt ly phoma lines (Raji) and chronic lymphocytic (B cell) leukemia as well as alloreactive T cell lines and Tcell leukemia lines (Jurkat, Molt-4) .

Cell lines derived from breast, lung and prostate carcinoma, and neuroglioma did not express NDA5. The cell membrane distribution of NDA5 indicates that this antigen is expressed only by cells of haematopoetic origin.

Example 4

Immunoprecipitation Studies of NDA5

NP-40 lysates of Raji cells, biosynthetically labeled

35 with S-methionine, were precipated with MόAb NDA5. The antibody precipitated under non-reducing conditions one band of 180kD. After reduction with 2- mercaptoethanol two distinct bands of approximately 85 and 75kD were distinguished. Example 5

Functional Studies of MoAb NDA5

The helper function of T-lymphocytes is generally studied in the Pokeweed Mitogen-system. Stimulation of B cells with PwM fails to induce blastogenesis and antibody secretion, unless T lymphocytes are added to the cultures. In a first series of experiments in which PBL from normal individuals were tested for their reactivity to PwM, the response was significantly higher when MoAb NDA5 was added at the initiation of the cultures. The results of these experiments are shown in Table 1 below.

Table 1

Responder MoAb NDA5 (5ug/ml) Day 3-Reactivity (mean cpm) 3H-TdR incorporation in cultures with:

Medium only PwM

No 210 12,394 Yes 470 33,088

NO 576 27,361 Yes 643 53,949

NO 213 52,270 Yes 500 91,875

No 1,957 36,380 Yes 1,326 71,473 Example 6

Determination of the Cause of MoAb NDA5's Enhancing Effect

It was next determined whether this enhancing effect results from- binding of the antibody to the T cell or to the B-cell component of the culture. For this, T and B lymphocytes were fractionated and purified from monocyte-depleted peripheral blood lymphocytes and tested for their response to PwM after one hour of incubation in medium containing MoAb NDA5 (5ug/ml) . Following the incubations the cells were extensively washed and tested for reactivity in cultures containing T cells alone, B cells alone or mixtures of T and B cells. The results of this experiment are shown in Table 2.

Table 2

Responding cells

T cells not preincubated (NP) 56 1,486

T cells preincubated (P) 68 2,436

B cells not preincubated (NP) 773 3,880

B cells preincubated (P) 141 2,136

NP + a 426 12,181

^TP + ^BNP 493 17,544

^TNP + ^BP 246 16,481

^Tp + ^Bp 585 24,295 These experiments showed that MoAb NDA5 has no mitogenic effect on either T or B lymphocytes and that there was only minimal reactivity to PwM when T cells (10,000 cells per culture) or B eels (50,000 cells per culture) were tested for PwM-reactivity alone rather than in mixtures. This low level of reactivity remained unchanged if the responding cells were preincubated with MoAb NDA5 prior to testing, when T and B cells were tested for PwM responsiveness together.

In mixtures contai .ni,ng 1 x 104T cells and 5 x 104B cells, pre-treatment of either components with MoAb NDA5 resulted in increased blastogenesis. The highest reactivity occurred when T cells and B cells were preincubated with MoAb NDA5 and then mixed and stimulated with PwM. These experiments suggest that MoAb NDA5 acts on molecules involved in the T-B interaction.

Example 7

Effect of MoAb NDA5 on Isolated T and B Cells

To determine whether MoAb NDA5 also increases the interaction of cells within the same compartment, and amplifies their reacivity, its effect was tested on purified T and B cells which were stimulated in vitro with PMA and with Staphylococcus Aureus Cowan Strain. The data from this experiment are shown in Table 3 below. Table 3

T cell and B cell reactivity to specific mitogens in the presence of MoAb NDA5

Responding Stimulus MoAb Reactivity 3H - TdR cells NDA5 (mean cpm) (5 ug/ml)

48 hours 72 hours

T cells PMA NONE

B cells SAC NONE

Responding T lymphocytes depleted of B cells and monocytes showed a significant increase in reactivity to PMA when tested in the presence of MoAb NDA5. No such effect was observed in the presence of equivalent amounts of other murine IgM antibodies used as isotype controls. Similarly, there was a sharp rise of B cell reactivity to SAC in the presence of MoAb NDA5. Taken together these data indicate that MoAb NDA5 is an efficient potentiator of both T and B cell reactivity.

Example 8

Determination of Cell Phases at which Lymphocyte Responses are Stimulated by MoAb NDA5

To determine whether this amplification effect is restricted to cells in early phases of activation or whether it is also observed when the antibodies added are to T or B lymphoblasts, two types of experiments were performed. First, the effect of MoAb NDA5 on T and B-cell proliferation was analyzed in cultures to which the antibody was added at various times after stimulation with PMA or with SAC, respectively. The potentiation of blastogenic responses occured only when cells were exposed to Mo b NDA5 within the first 24 hours of stimulation. Mo b NDA5 did not sustain the blastogenic response of activated T or B lymphoblasts after the peak of reactivity had occurred. There was a shift of the peak to the left, i.e. the blastogenic response was both increased and accelerated when the antibody was added at the time when the cultures were initiated. In contrast, addition of the antibody to the cultures 48 hours after activation with SAC or with PMA had little effect, if any. Second, the effect of MoAb on the growth and rate of 3H-TdR incorporation in leukemic cells of T cell origin (MOLTA and Coleman- leukemia) and of EBV-transformed B cell lines was tested. MoAb NDA5, in contrast to the previously described MoAb NDA. and NDA., did not amplify the proliferation and maturation of B lymphoblastoid cell lines, and failed to influence the growth of T leukemias.

Thus, these studies in addition to the lack of stimulatory effects of MoAb NDA5 on malignant and transformed lymphoblasts suggest that MoAb NDA5 acts on cell surface structure(s) associated with activation rather than with growth.

Example 9

The Effects of MoAb NDA5 on Immune Responses in T Lymphocytes Primed In Vivo and In Vitro

To determine whether specific immune responses exhibited by T lymphocytes primed in vivo and in vitro are also exacerbated by MoAb NDA5, these two systems were analyzed. In the first system, the reactivity to Tetanus Toxoid was tested in lymphocytes from patients who have been recently vaccinated against Tetanus. In the presence of MoAb NDA5 there was an early and significant increase of reactivity to optimal and suboptimal concentrations of Tetanus Toxoid.

In the second system, T lymphocytes were primed to allogeneic stimulating cells in mixed lymphocytes cultures, in the presence and absence of MoAb NDA5. As expected, the blastogenic response, measured on days 5 and 6, was significantly higher in the presence of MoAb NDA5 than in cultures either with control antibodies or without any murine Ig. These primary cultures were tested until day 10, and were then rechallenged either with the specific (original) stimulator or with irradiated PBL from individuals sharing no HLA-D/DR antigen with the specific stimulator. The memory response of the secondary MLC was measured after 48 hours by quantiating the rate of 3H-TdR incorporation. Data from these experiments are shown in Table 4 below.

Table 4

Effect of MoAb NDA5 on l m hoc te reactivit to

The 48 hour response to the specific stimulator was greatly enhanced when the primary cultures were generated in the presence of MoAb NDA5 and when this antibody was added at the time of secondary stimulation to cultures grown without MoAb NDA5 during the first 10 days of primary MLC activation. These data indicate that MoAb NDA5 potentiates significant memory immune responses induced in vivo by specific soluble antigens or induced in vitro by allogeneic HLA-D/DR antigens. Thus, MoAb NDA5 may represent a potent adjuvant for in vivo or in vitro sensitization of T lymphocytes.

Example 10

To determine whether the reactivity of T lymphocytes from patients whose immune response is compromised by HIV or leukemia can also be increased, the effect of MoAb NDA5 on the PwM-response of PBL from AIDS and leukemic patients was tested.

There was a dose-related increase of T cell reactivity when MoAb NDA5 was added to cultures of PBL from AIDS patients. Both the earliness of the response and the degree of augmentation were indicative of an effective restoration of reactively normal blastogenesis. Similarly, PBL from patients with CLL and myelogenous leukemia showed enhanced reactivity in the presence of MoAb NDA5. The data from this experiment are shown in Table 5 below.

Table 5

Amplification of T cell responses to PWM in healthy individuals and patients with

AIDS, CLL or Myelogenous Leukemia

Concentration of Day 3 Day 5

Responder MoAb NDA5 (ug/wl) Reactivity: 3H-TdR(mean cpm)

Normal 0 23,580 29,506

1 52,057 71,498

2 55,557 69,539

5 53,949 69,292

10 52,945 62,991

AIDS 0 12,394 16,344

0.125 13,895 21,534

0.250 15,381 23,531

0.500 18,508 41,781

1 20,174 52,154

2 23,249 53,514

5 26,053 52,492

10 25,050 60,862

CLL 0 1,656 3,979

5 3,896 16,666 M.L. 0 5,670 8,945

5 11,595 401

Claims

What is claimed is:

1. A purified, differentiation antigen designated NDA5 associated with amplification of the proliferative responses of T and peripheral B lymphocytes to mitogens and antigens, characterized by being a heterodim4er having an apparent molecular weight under non-reducing conditions of about 170-180 kd, by being comprised of two subunits having apparent molecular weights of about 75kd and 85kd, and by being im unologically reactive with the monoclonal antibody designated MoAb NDA5 (ATCC Accession No. HB9848) .

2. A purified, human differentiation antigen of claim 1 which is a non-lineage-specific, leukocyte- common antigen (LCA) .

3. A purified, human differentiation antigen of claim l.

4. An antibody ^' directed to, and capable of specifically forming a complex with, the purified differentiation antigen of claim 1.

5. A monoclonal antibody of claim 4.

6. The monoclonal antibody designated MoAb NDA5 (ATCC Accession No. HB9848) .

7. A hybridoma which produces a monoclonal antibody of claim 5.

8. The hybridoma ATCC Accession No. HB9848.

9. A method of augmenting the responsiveness of T and peripheral B lymphocytes to a mitogen and an anti¬ gen which comprises contacting T and peripheral B lymphocytes under suitable conditions with an antibody of claim 4 or 6 in the presence of the mitogen or antigen so as to effect an augmented response by the T and peripheral B lymphocytes.

10. A method of claim 9, wherein T and peripheral B lymphocytes are human lymphocytes.

11. A method of claim 10, wherein lymphocytes are peripheral B lymphocytes from a patient whose immune response is compromised.

12. A method of claim 11, wherein the patient is infected by HIV, has chronic lymphocytic leukemia, myelogenous leukemia, or multiple myeloma.

13. A method of claim 10, wherein lymphocytes are T lymphocytes from a patient whose immune system is compromised.

14. A method of claim 13, wherein the patient is infected by HIV, or has chronic lymphocytic leukemia, myelogenous leukemia, or multiple myeloma.

15. A method of claim 9, wherein the antigen comprises a soluble antigen and allogenic stimulator of primary and secondary mixed lymphocyte cultures.

16. A method of claim 15, wherein the soluble antigen comprises Purified Protein Derivative of Microbacterium Tuberculosis or Tetanus Toxoid.

17. A method of potentiating an immune response to a specific antigen which comprises contacting T or peripheral B lymphocytes with a mixture comprising the specific antigen and an antibody of claim 4 or 6 in an amount effective to potentiate the immune response.

18. A method of claim 17, wherein T or peripheral B lymphocytes are human T or peripheral B lymphocytes.