By this time those who do not have experience of the task of obtaining funds for research may be wondering what a grant application actually looks like. Basically, the forms are all the same and I show below some sections of an application I submitted (30 copies) to the US National Institutes of Health in May 1977. There are strict limits on the number of pages, and most space is allotted for the applicant's proposal, rather than the applicant's track record.

    The first page contains the proposal summary (title of the project, the applicant's institutional address, and the signatures of the applicant and a senior member of the institution; e.g. the Dean of Medicine).

    The second page contains a Table of Contents with page numbers:

PROJECT TITLE

Immunotherapy: 

New Approaches to Immunotherapy:

Extra-Corporeal Clonal Expansion Therapy (ECCET)

In the budget I asked for $168,285 over 3 years. This included research materials ($43,650), a research assistant with a Ph.D ($45,000), a technician with a B.Sc ($30,000) and pension contributions, etc., for these ($7,500). For equipment I requested an automatic harvester of cell cultures ($3,300). There was also a 30% overhead to cover institutional costs ($38,835).

4. BRIEF SUMMARY OF PROPOSAL

There are reasons to believe that cancer patients possess a few lymphocytes bearing receptors of moderate specificity for tumor-specific antigenic determinants. However, under in vivo conditions encounter of these cells with tumor determinants leads to lymphocyte destruction (tolerance) rather than lymphocyte proliferation. Thus clones of lymphocytes bearing receptors of moderate specificity cannot expand in vivo. If cultured in vitro with tumor antigenic determinants the clones would be able to expand, provided the tolerance induction process were switched off, and culture conditions were favourable for cell proliferation. Our laboratory has presented the first in vitro evidence that complement is involved in the switching off (clonal deletion) of lymphocytes by specific antigen. Thus if cells are cultured under conditions designed to eliminate or reduce the action of complement, tolerance induction can be prevented. Lymphocyte clones of moderate specificity for tumor determinants can thus expand free from in vivo constraints. Infusion of such expanded autologous clones back into the patient at a rate sufficient to overwhelm the tumor could then be feasible.

5. INTRODUCTION AND BACKGROUND

This proposal falls partly under the area of RFP 74144-31, entitled "Immunization with Autochthonous Tumor". However, there are novel (and hence controversial) elements to the present approach and thus I am submitting the proposal under the heading of RFP 74141-31, "Immunotherapy: New Approaches to Immunotherapy" For a general introduction to the background in this area see Trentin (1976).

(a) ECCET begins with the removal of lymphoid cells from a patient with a neoplasm. This may be: 

• (i) a few ml of blood from which lymphocytes may be purified (Boyum,1968), or 
  
  
• (ii) selective removal of blood lymphocytes by continuous flow leukapheresis (Pentycross, Walden & Bagshawe,1976), or 
  
  
• (iii) removal of a lymph-node from which lymphoid cell suspensions can be prepared.

(b) ECCET ends with the transfer from the laboratory to the clinic of one or more 500 ml blood transfusion bottles each containing some 10¹⁰ autologous immunologically competent cells each bearing receptors of a moderate specificity for specific antigenic determinants borne by the tumor. The patient is then transfused with the autologous lymphocytes which react specifically with tumor cells and destroy them.

(c) The essence of this RFP response is a description of the procedure by which lymphocytes of moderate specificity for the surface determinants of a tumor may be prepared in large numbers from a patient. Recent developments in this laboratory have provided an understanding of the culture conditions needed for a proliferative, rather than a tolerogenic, lymphocyte response to a given antigenic determinant. Complement appears to be needed for the tolerogenic response (Azar & Good,1971a,b; Forsdyke, 1973e,f). Thus cells must be cultured under conditions devised to reduce or eliminate the action of complement. This involves using 

• (i) serum heated at 58^oC for 20 minutes to destroy certain heat-labile complement components (Forsdyke,1973e,f) and
   
  
• (ii) 2-mercaptoethanol (2-ME) which appears to protect the cell membrane against attack by complement (Forsdyke,1977c,g).

(d) In theory a tumor will have eliminated all those immunologically competent cells bearing receptors of high specificity for tumor-specific antigenic determinants. Such determinants will be read as "self" and an immune response will not be mounted against them. Figure 1 shows the expected distribution of the specificities of lymphocytes from a patient with an established tumor(Forsdyke 1975a).

Cells of high specificity for tumor-specific antigenic determinants will have been eliminated. Most cells of moderate specificity will also have been eliminated. However there will be present in the patient a very few cells belonging to moderate specificity clones (hatched area in Fig.1). Under in vivo conditions moderate specificity clones will not be able to expand. The response of cells from such clones to encounter with tumor-specific antigenic determinants in vivo will be cell destruction rather than cell proliferation. The aim of ECCET is to obtain such cells and culture them under in vitro conditions such that clonal expansion rather than clonal deletion can occur.

Infusion of moderate specificity lymphocytes would not be so effective as the infusion of high specificity lymphocytes. However the difference should be partly overcome by quantitative factors. 1000 moderate specificity lymphocytes might be as effective as 1 high specificity lymphocyte. Provided moderate specificity lymphocytes can be prepared in large numbers the problem of the degree of specificity should be overcome.

6. EXPERIMENTAL PLAN

ECCET could be applied now to the treatment of cancer patients. The technology is in place. The ideas are in place. The limiting factor is acceptance of the ideas. If clonal deletion requires the complement dependent destruction of cells by antigenic determinants (Burnet,1959; Azar & Good 1971 a,b; Forsdyke,1969a, 1975a) then ECCET should work. The prime task at this time is to provide convincing evidence for or against the clonal deletion hypothesis. Thus in the 3 year time period of the present proposal I intend to emphasize this aspect of the work. Studies with animal model systems will be continued (forsdyke,1973e,f; 1977b,c,d). Key experimental results will be confirmed using periferal blood lymphocytes from healthy human volunteers. These volunteers will be immunized with fresh antigens or their past immunological history will be taken into account when deciding which antigen to use in challenging their cells. Dose-response curves for cell proliferation in vitro will be determined. The cultures will contain autologous serum preheated at 56°C-58°C and and 2ME. (The concentration of 2ME must be adjusted according to the serum concentration and the temperature of preheating serum; Forsdyke,1977d,g) The lowest antigen concentration capable of producing cell proliferation will be determined. Cultures containing such antigen concentrations (presumed to have stimulated. the cells bearing high specificity receptors) will be maintained with medium replenishment for a time-period sufficient to allow high specificity clones to selectively expand. Such clones should give an approximately unimodal dose-response curve to antigen (Fig. 2a) with a more dramatic inhibition at high antigen concentrations than I have been able to achieve to date (Forsdyke,1973e;1977c,d; see Fig.2b).

We will then attempt to demonstrate that the inhibition is complement dependent. We will purify or obtain commercially, or borrow, purified complement components (e.g. factor B of the properdin pathway). These will be added to cultures containing heated serum in order to demonstrate that complement is the serum activity responsible for the inhibitory effect of unheated serum at high antigen concentrations.

Other means of selecting and expanding high specificity clones which will be considered will be (i) the use of agar plate cultures and low antigen concentrations to try to obtain plaques of high specificity cells (Metcalf et al., 1975) and (ii) the addition of lectins to cultures to stimulate cell proliferation after the specific cells have been selected (Frenster,1976). The essence of the problem is devising the right culture conditions. Under optimum conditions cells should proliferate exponentially to produce cells of the desired specificity in unlimited quantities.

If the above described experiments are successful we will be in a position to culture the peripheral blood lymphocytes of cancer patients with their own tumor specific antigens. The tumor specific antigens may be presented to autologous lymphocytes as 

• (i) intact tumor cells (Golub, l977), 
  
  
• (ii) crude homogenates of tumor cells, or 
  
  
• (iii) membrane fractions from tumor cells. 

The problem of purifying tumor-specific antigens is one to which the NIH giving much attention and will not concern us at this stage.

Major factors supporting my research proposal are  (i) my dual qualifications in medicine and biochemistry (see attached curriculum vitae), (ii) my extensive experience in lymphocyte culture and nucleic acid research (see attached list of publications), (iii) my position in a biochemistry department in a Faculty of Medicine at a university which is closely located to clinical facilities (my laboratory is 100 yards from the Kingston General Hospital which is the major teaching hospital of the Faculty of Medicine.

REFERENCES

Azar,M.M. & Good,R.A. (1971a) J. Immuno1.106, 2241. The inhibitory effect of vitamin A on complement levels and tolerance induction.

Azar,M.M. & Good,R.A. (1971b) Proc.Soc.Exp.Biol.Med.137, 4229-432. Effect of fumaropimaric acid on mouse complement and immunological tolerance.

Boyum,A. (1968) Scand.J.C1in.Lab.Invest.21 (suppliment 97),1-119. Isolation of mononuclear cells and granulocytes from human blood.

Burnet,F.M. (1959) The Clonal Selection of Acquired Immunity. Cambridge University Press. p.64.

Forsdyke,D.R. and coworkers. (Please see list of publications)

Frenster,J.H. (1976) Annals.N.Y.Acad.Sci.277,45-51. "PHA-activated autochthonous lymphocytes for systemic immunotherapy of human neoplasms."

Golub,S.H. (1977) Ce11..Immunol..28, 379-389. In vitro sensitization of human 1ymphoid ce11s to antigens on cultured melanoma cells. II. Sensitization against melanoma antigens.

Metcalf,D.,Nossa.1,G.J.V.,Warner,N.L.,Miller,J.F.A.P. Mandel,T.E.,Layton,J.E. & Gutman,G.A. (1975) J.Exp.Med.142,1534-1549. Growth of B-Lymphocyte colonies in vitro.

Pentycross,C.R., Walden,P.A.M. & Bagshawe,K.D. (1976). Vox.Sang,31,446-450. Collection of Lymphocytes in a Continuous Flow Blood Cell Separator.

Trentin,J.J. (1976) Annals.N.Y.Acad.Sci.277,716-721. "Tumor Immunotherapy in Experimental Animals. Current Status and Prospectus."

(For further background references please see references in my published work and in volume 277 of Annals of N.Y.Acad.Sci.)

Gentlemen:

This is to advise you that the appropriate technical review committee of the National Cancer Institute has completed its evaluation of all proposals received for the project referenced above. Although an award has not been made to date, I felt you would like to know as soon as possible that your proposal did not receive a rating sufficiently high to place it within the zone of final consideration.

We thank you for your interest in this program and hope that there will be future opportunities for you to participate in the Tumor Immunology Program of the National Cancer Institute.

Sincerely yours,

Robert S. Townsend, Contracting Officer, 
Research Contracts Branch, National Cancer Institute

cc: Dr. Donald R. Forsdyke

Fortunately, the NCI was able to fund others to continue this line of work, and a quarter of a century later it is still the focus of intensive effort.

D. R. Forsdyke March 2004

For an example see Dijkstra et al. (2018) Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174:1586-98.

D. R. Forsdyke Sept 2018

ECCET simply required a blood from the cancer patient. Steve Rosenberg's group used "tumour infiltrating lymphocytes" (TILs), which required a tumour sample. But tumours mutate and spread, so that their antigenicity is no necessarily stationary. Blood lymphocytes are more likely to have a range of antigen specificities among which some may address, both a primary cancer locus with its mutations, and secondary loci with their mutations. Thus, the very last cancer cell should be eliminated. However, success of a modified TIL form of ECCET was reported by K. Garber in Nature Biotechnology (Aug 7, 2019): 

"Earlier in the summer, at the annual meeting of the American Society of Clinical Oncology (ASCO), surgical oncologist Stephanie Goff of the US National Cancer Institute (NCI) presented a case report of a 49-year-old woman with advanced breast cancer who was treated with her own T cells, obtained from the tumor. After the cells were expanded ex vivo, the patient was infused with 80 billion cells. Her tumors completely disappeared, and she has remained tumor-free for 42 months. 'TILs are capable of eliminating the last cancer cell,' Goff said at ASCO."

D. R. Forsdyke Sept 2019