Published in: "Proceedings of the Fifth Leukocyte Culture Conference", (Harris J, ed.), pp. 359-373 (1970), New York: Academic Press. Presented at the 10th International Cancer Congress, Houston, Texas, May, 1970.

Department of Medicine, Stanford University School of Medicine, Stanford, California 94305

Introduction:
Lymphocytes in Human Neoplasms:
Human Tumor-Associated Antigens:
Immune Responses to Human Neoplasms:
Activation of Lymphocytes from Cancer Patients:
Clinical Use of Autologous Lymphocytes after In-Vitro Activation:
Limitations of Clinical Immunotherapy:
Biological Implications:
Summary:
Support:
References:
Conference Discussion:
Additional References:
Links:

INTRODUCTION

Clinical therapy of disseminated neoplasms in man is limited by (1) the selective affinity of the therapeutic agent for the neoplastic cells compared to the normal cells (Goldin, 1969), and (2) by the relative vulnerability of the neoplastic cells compared to the normal cells to the agent (Sartorelli, 1969). Current chemotherapeutic agents generally have a low selective affinity for neoplastic cells in-vivo in man (Heidelberger, 1969). Favorable clinical chemotherapeutic effects seem to require that the neoplastic cells have higher rates of cell division or of cell biosynthesis than normal cells in-vivo (Hart, et al, 1969). When such is not the case, the neoplastic cells are generally invulnerable to toxic in-vivo levels of the drug (Schabel, 1969). A potential advantage of immunotherapeutic agents is that they could be expected to display a high selective affinity for the neoplastic cells, and might display their cytotoxicity against those neoplastic cells which usually would be invulnerable to chemotherapeutic agents because of their low rates of cell division or of cell biosynthesis.

LYMPHOCYTES IN HUMAN NEOPLASMS

Pathologists have long noted the histologic infiltration of lymphocytes into neoplasms removed from untreated patients. With the recognition of the immunologic role of the lymphocyte, it became possible to envision such lymphocytic infiltration as a host immunologic response to the neoplastic cells, in a pattern similar to that seen in the homograft rejection reaction (Murphy, 1926). Subsequent clinical-pathologic correlation studies have revealed a close and direct relation between the intensity of lymphocytic infiltration within an untreated human neoplasm and the subsequent life-survival of the particular patient. These correlations have been demonstrated for such diverse human neoplasms as testicular seminoma (Dixon and Moore, 1953), gastric carcinoma (Black, et al, 1956), breast carcinoma (Hamlin, 1968), Hodgkin's Disease (Keller, et al, 1968), and neuroblastoma of infancy (Martin and Beckwith, 1968). These correlation results have emphasized the necessity of re-examining the questions of the antigenicity of human neoplasms and of the immune status of cancer patients.

HUMAN TUMOR-ASSOCIATED ANTIGENS

One of the most significant advances in our understanding of the biology of human neoplasms has been the recent demonstrations that a wide variety of human neoplastic cells possess tumor-characteristic antigens on their cell surfaces. With the isolation and purification of such neoplastic cell antigens, it has become possible to study both the clinical and the embryologic time-correlates and quantity-variations of these antigens. Human neoplasms from which tumor-associated antigens have been isolated are listed in Table 1.

Indirect evidence for the presence of such antigens is available for a wide variety of other human neoplasms (Hellstrom, et al, 1968).

Embryologic studies have revealed that human tumor-associated antigens are often identical to antigens present on normal embryonic cells (Gold and Freedman, 1965b) but not normally present on adult cells of the organ system under analysis (Gold and Freedman, 1965a, b). Such neoplastic-embryologic correlations within single organ systems illustrate the widespread occurrence of the phenomena of de-repression of fetal genes as a characteristic molecular mechanism in human neoplastic cells (Frenster, 1970), but do not as yet prove that such gene de-repression is responsible for the neoplastic behavior of these cells (Frenster, 1965, 1966).

Purification of tumor-associated antigens has also permitted the development of radio-immunoassays of circulating tumor antigens in the blood of tumor-bearing patients (Thomson, et al, 1969). The titers of such antigens are often inversely related to the titers of free antibodies specific for such antigens (Gold, 1967), suggesting the in-vivo formation of antigen-antibody complexes in advanced cancer with antigen excess (Thomson, et al, 1969). Assays of circulating free tumor antigen have been found to closely reflect the clinical status and progression of the neoplasm, especially in the post-operative period following resection of the primary neoplasm (Thomson, et al, 1969).

IMMUNE RESPONSES TO HUMAN NEOPLASMS

With the isolation and purification of tumor-associated antigens from diverse human neoplasms (Table 1), it has become possible to devise clinical assays for: (1) complement-fixing cytotoxic antibodies (Gold, 1967; Eilber and Morton, 1970), (2) sensitized circulating immune lymphocytes (Jehn, et al, 1970), and (3) skin-reactivity to the tumor-associated antigens (Fass, et al, 1970a, b; Hollinshead, et al, 1970). In addition, with the ability to grow human tumor cells in-vitro (Hellstrom, et al, 1968), it has become possible to clinically assay for: (1) non-complement-fixing blocking or enhancing antibodies (Hellstrom, et al, 1969), (2) cytotoxic antibodies (Hellstrom, et al, 1968), and (3) cytotoxic circulating lymphocytes (Hellstrom, et al, 1968). Where adequate studies have been completed, the available data suggest that individual patients respond to the presence of a neoplasm by the formation of: (1) circulating cytotoxic lymphocytes specifically sensitized to the tumor-associated antigens Hellstrom, et al, 1968), (2) tumor-specific complement-fixing cytotoxic antibodies (Gold, 1967; Hellstrom, et al, 1968; Eilber and Morton, 1970), and (3) tumor-specific blocking or enhancing antibodies (Hellstrom, et al, 1969). The latter type of enhancing antibodies, and the presence of elevated titers of free cirulating tumor antigen in patients with recurrent or widespread neoplasms (Thomson, et al, 1969), are two mechanisms which result in a reduced effectiveness of the immune response in containing the neoplasm. Other mechanisms reducing the effectiveness of the immune response are: (1) inadvertant immunosuppresion during radio-therapy or chemotherapy of the neoplasm (McKhann, 1969; Mitchell, et al, 1969), (2) immuno-selection of less antigenic neoplastic cells during the formation of metastases (Davidsohn and Ni, 1970), and (3) possible modulation of tumor cell antigenicity in the face of the immune response (Davidsohn and Ni, 1970).

ACTIVATION OF LYMPHOCYTES FROM CANCER PATIENTS

Since antibody responses to human neoplasms can include the formation of deleterious blocking or enhancing antibodies (Hellstrom, et al, 1969), our immunotherapy program has concentrated on the lymphocyte-mediated aspects of immunity to human tumors (Frenster and Rogoway, 1968). Such lymphocyte-mediated immunity has been demonstrated in a wide variety of human neoplasms (Hellstrom, et al, 1968), and these immune lymphocytes can undergo a significant increase in their immune cytotoxic activity against human neoplastic cells in-vitro if the lymphocytes are first activated by such mitogens as phytohemagglutinin (Chu, et al, 1967). Lymphocyte immunity against neoplasms appears to be mediated by lymphocytes which are thymus-dependent (Miller and Osoba, 1967), and such lymphocytes are preferentially activated by phytohemagglutinin (Davies, et al, 1968; Greaves, et al, 1968). Other mitogenic agents such as pokeweed mitogen appear to activate separate, possibly non-thymus-dependent, lymphocyte populations (Brown, et al, 1970), while such lymphocyte mitogens as concanavalin and anti-lymphocyte serum inhibit the cytotoxicity expressed by immune lymphocytes while allowing activation of the lymphocytes to proceed (Perlmann, et al, 1970).

Direct apposition of the immune lymphocyte against the neoplastic cell appears to be required for maximal cytotoxicity (Biberfeld, et al, 1968; Archibald and Frenster, 1970), although soluble cytotoxic factors (Kolb and Granger, 1968) and anti-mitotic factors (Cooperband, et al, 1970) are also released by immune lymphocytes during activation by phytohemagglutinin. Although relatively pure preparations of phytohemagglutinin have been injected into patients with advanced neoplasms ((Robinson, 1966), the rapid development of antibodies to such phytohemagglutinin preparations (Simons, et al, 1968) has prevented their long-term use.

Because of these limitations, we developed a method for the large-scale in-vitro activation of lymphocytes from patients with advanced neoplasms, with the subsequent re-infusion of such activated lymphocytes into the original donor for immunotherapeutic purposes (Frenster and Rogoway, 1968). In the most recent modification of this procedure, hospitalized patients with advanced cancer receiving no other form of therapy donate 10 ml/kg of whole blood into heparinized Fenwal donor plastic bags under sterile procedure throughout. The leukocyte-rich supernate is transferred to a second bag after initial centrifugation at 20 g for 10 minutes, and the separated erythrocytes are are re-infused into the original donor patient within one hour of the original donation. The leukocyte-rich supernate is then centrifuged at 200 g for 10 minutes, and the seaparated cell pellet is washed three times in a closed system with 300 ml of a 1:4 sterile dilution of human serum albumin in medium 199 with 4 units of heparin/ml. The washed cells are then re-suspended in 500 ml of the same mixture of albumin-medium 199-heparin to which is added 10 ug/ml of the highly-purified glycoprotein octamer of phytohemagglutinin (Rigas and Head, 1969) and 100 mg each of penicillin and streptomycin. The cells are incubated in horizontal Fenwal bags at 35^oC. for 24 hours, the medium changed, and the incubation continued for asecond 24 hour period. The cells are then washed three times with 300 ml. of the sterile albumin-medium 199-heparin mixture, are re-suspended in 100 ml. of this mixture, and are then re-infused into the original donor patient at a rate of 10 ml/min.

Under these conditions, up to 10⁹ lymphocytes can be donated during each batch donation by the patient, and of these, approximately 30 percent in a viable and activated state can be re-infused into the patient following the in-vitro activation procedure (Frenster and Rogoway, 1968). The removal of the autologous plasma during the original leukocyte separation serves to remove any plasma factors (Silk, 1967) which might inhibit phytohemagglutinin activation of the lymphocytes, and also serves to remove all coagulation factors which otherwise might permir coagulation within the culture.

CLINICAL USE OF AUTOLOGOUS LYMPHOCYTES AFTER IN-VITRO ACTIVATION

The re-infusions of activated autologous lymphocytes are well tolerated, with no evidence to-date, two years since the first re-infusions, of any occurrence of hemolysis, leukopenia, leukemia, thrombocytopenia, thromboembolism, anaphalaxis, lympho-proliferative disease, or auto-immune disease. Immediately after each re-infusion, the patient usually experiences mild chills, followed within one hour by fever to 38.5^oC, but these side -effects can be reduced by pre-treatment of the patient with aspirin. Patients are usually hospitalized for one week each month, giving whole blood donations on days 1-5, and receiving re-infusions of activated lymphocytes on days 3-7. Patients are ambulatory after re-infusion of the donated erythrocytes or within 2 hours after re-infusion of the activated lymphocytes, and return to work or school one day after the last re-infusion.

Five consecutive asymptomatic patients with widely-disseminated neoplasms have been studied in sufficient detail to-date (May, 1970) (Table 2).

Each patient was selected because of the presence of multiple pulmonary metastases which could be quantitatively assessed by serial chest radiography. Each patient had a neoplastic histology which was judged to offer little or no potential for cure by chemotherapy; by virtue of the multiple pulmonary metastases, no curative appraoch by surgical or radiotherapy techniques was feasible. Because each patient was asymptomatic, the fully-informed consent of each patient was obtained before inclusion in the study. Each patient received daily re-infusions of more than 10⁸ activated autologous lymphocytes for 5 days over 1-6 courses without adverse effects (Frenster and Rogoway, 1970).

Of the five consecutive patients studied in sufficient detail to date (Table 2), three patients have shown significant objective regressions in pulmonary metastases of greater than 50 percent in the product of two dimensions of individual metastases lasting for longer than one month after re-infusion (Frenster and Rogoway, 1970). The responders included one patient with generalized malignant melanoma, one patient with metastatic Ewing's sarcoma, and one patient with metastatic embryonal cell carcinoma of the testis (Frenster and Rogoway, 1970). The remaining two patients, one with generalized malignant melanoma and one with metastatic teratocarcinoma of the testis have displayed neither tumor regressions nor tumor accelerations (Frenster and Rogoway, 1970). One patient's regression lasted for 3 months following the last re-infusion of activated lymphocytes. Each responding patient has shown heterogeneity of response among pulmonary nodules, with some nodules regressing to a greater degree than other nodules. Presumably, such heterogeneity of response reflects evolving heterogeneity of tumor clones composing each nodule, and/or unequal distribution of re-infused lymphocytes among the multiple nodules.

LIMITATIONS OF CLINICAL IMMUNOTHERAPY

Although each of our patients had relatively small pulmonary metastases (3 cm or smaller in diameter), none of the patients have received either limiting amounts or sufficient amounts of activated autologous lymphocytes. A pulmonary nodule 3 cm in diameter can contain up to 10⁹ neoplastic cells. We have been able to re-infuse 10⁸ activated lymphocytes per day for 5 consecutive days each month for 1-6 months in our study patients. At best, if all re-infused lymphocytes localized into one metastatic nodule, the lymphocyte/neoplastic cell ratio would approach 1.0, although it could be locally higher at the periphery of the nodule. In-vitro studies suggest that a lymphocyte/neoplastic cell ratio of 25.0 is probably necessary for optimal cytotoxicity against tumor cells over short periods of time (Perlmann, et al, 1968). In-vitro studies also suggest that lymphocytes survive the lethal immune encounter with target cells (Perlmann, et al, 1968), so that over a longer period of time, one lymphocyte might destroy several neoplastic cells serially. In addition, it is possible that each activated lymphocyte which is re-infused might recruit other lymphocytes, macrophages, and/or plasma cells within the patient by transfer of the activated state (Roitt, et al, 1969). The duration of persistance in the activated state is not known for activated lymphocytes in-vivo, although in-vitro this may not exceed 14 days in the absence of continued mitogenic stimulation (Polgar, et al, 1968). The in-vivo influence of blocking antibodies (Hellstrom, et al, 1969) and of circulating tumor antigens (Thomson, et al, 1969) on cytotoxic reactions mediated by activated immune lymphocytes against autochthonous neoplastic cells cannot be assess at this time. The distribution and penetration into neoplasms after re-infusion of activated lymphocytes is currently under study.

It is conceivable that larger numbers of lymphocytes could be re-infused after collection by continuous-flow blood cell separation (Judson, et al, 1968), or after 1-year proliferative lymphocyte culture in-vitro (Moore and Moore, 1969). Other approaches include employing agents such as BCG for repeated non-specific in-vivo immune activation (Mathe, et al, 1969). Since the mitogenicity of the purified glycoprotein octamer of phytohemagglutinin resides in two of the eight monomer subunits (Rigas and Head, 1969), it is possible that in-vivo use of such mitogenic subunits would result in significant in-vivo lymphocyte activation without the simultaneous induction of antibodies directed against these smaller and less particulate subunits. Such studies are currently in progress.

In patients with advancing neoplasms, both the number ofneoplastic cells and thetiter of circulating tumor antigens increases (Thomson, et al, 1969). Both of these increases are unfavorable for successful lymphocyte immunotherapy. It may therefore be necessary in patients with advancing disease or bulky disease to first employ surgery, radiotherapy, and/or chemotherapy to reduce the number of tumor cells and the titer of circulating tumor antigen before lymphocyte immunotherapy can be employed with maximal effectiveness, even though radiotherapy and/or chemotherapy can themselves result in temporary immuno-suppression (Mitchell, et al, 1969). In this regard, protocol studies are now under way in which surgical patients are randomized to include post-operative lymphocyte immunotherapy, in order to assess the possible value of adjuvant immunotherapy after the neoplastic mass has been reduced by surgical resection of the primary site.

BIOLOGICAL IMPLICATIONS

It is possibly of some importance to our eventual understanding of both the neoplastic process and the immuno-therapeutic process that de-repression of normally repressed host genes is found to occur within both processes (Frenster, 1970). Thus, in a large variety of untreated human neoplasms, tumor-associated antigens appear via de-repression of normal fetal antigens which are usually not expressed in adult life (Table 1). In addition, normally-repressed host species of nuclear RNA are regularly synthesized within human leukemic cells (Neiman and Henry, 1969), while the production of ectopic polypeptide hormones by neoplasms of non-endocrine tissues has similarly been interpreted as examples of gene de-repression within human neoplasms (Omenn, 1970).

During the course of in-vitro lymphocyte activation, the highly-purified glycoprotein octamer of phytohemagglutinin penetrates into the nucleus of the lymphocytes and is bound preferentially to hetrochromatin complexes (Stanley, et al, 1968, 1970), where it effects a conversion of heterochromatin to active euchromatin (Tokuyasu, et al, 1968). Such nuclear binding of this activating ligand is rapidly followed by de-repression of lymphocyte RNA synthesis (Cooper, 1968) and the release of lymphocyte cytotoxins (Kolb and Granger, 1968) as outlined in Table 3.

Current hypotheses of oncogenesis in mammalian systems emphasize the importance of gene de-repression mechanisms within potententially-oncogenic genes (Huebner and Todaro, 1969). Such gene de-repression within neoplastic cells may similarly involve the nuclear binding of oncogenic activating ligands (Frenster, 1965), in which case the parallel phenomena of gene de-repression within human neoplasms and within immuno-therapeutic lymphocytes (Table 3) could be of importance to studies of both cancer therapy and cancer prevention.

SUMMARY

Now that a wide variety of human neoplasms have been shown to possess tumor-associated antigens to which the patient responds with lymphocyte-mediated immunity, it has become both possible and necessary to develop immnuo-therapeutic approaches which can take advantage of these phenomena for the benefit of the cancer patient. One approach to such immunotherapy appears to be the use of autologous lymphocytes from each cancer patient, to whom the lymphocytes can be returned with safety and some effectiveness after in-vitro activation. It now seems necessary to effect such lymphocyte activation in-vivo within each patient.

SUPPORT

Supported in part by NIH research grants CA-10174 and AM-01006, and by a Research Scholar Award from the Leukemia Society to Dr. Frenster.

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Brostoff (London): Wouldn't it be easier to inject PHA intravenously?

Frenster (Stanford): There are several studies (Gamble CN, (1966), Blood 28: 175; Simons et al, (1968), Nature 219:1021) which suggest that there is a rapid development of antibodies to the PHA and the presumption has been that such antibody produced inactivates the injected PHA. Robinson has studied the parenteral use of PHA in terminal cancer patients and does report minimal clinical responses, but the time course available for such administration is limited by the development of such antibodies to PHA (Robinson E, (1966), Lancet 2: 753).

Falk (Toronto): All the work that I am aware of on in-vitro cytotoxicity by PHA stimulated lymphocytes suggests that this reaction is entirely nonspecific. Why do you feel that giving patients lymphocytes that have been triggered with PHA are going to go to the cancer any more than the other tissues?

Frenster (Stanford): We know that there are significant numbers of lymphocytes that are already specifically sensitized for the tumor antigen (Hellstrom I, et al, (1968), Nature 220: 1352) and it is those particular lymphocytes that we are hoping to influence by the in-vitro activation.

Falk (Toronto): Well, wouldn't it be better to culture these lymphocytes with the actual tumor, then you specifically sensitize them or specifically trigger them further to the tumor antigen? If there is an inhibitory factor in the serum or an enhancing antibody you could culture them in normal serum.

Frenster (Stanford): We don't always have access to the tumor for one thing., and we are not sure that the tumor antigens would be as efficient a mitogenic stimulus as PHA is. Granted, we probably lose some specificity in the process (Chu EHU, et al, (1967), J. Natl. Cancer Inst. 39: 595).

Stewart (Ottawa): Complimentary to this, we've studied some 170 patients in the course of skin testing with extracts of the primary and quite frequently the metastatic tumor and graded these tumors in terms of stromal infiltrate of mono-nuclear cells. Approximately 70% have insignificant stromal infiltrate. Only about 30% have it and really I was going to ask the same question - can you demonstrate that you are encouraging lymphocytes to go in initially? This would be very exciting but I would predict that in probably 70% of the patients you would never get them in there, for some reason or another.

Frenster (Stanford): Well, I think we are getting them in. We have parallel studies in which we are tracing in-vivo the lymphocytes that have been labelled in-vitro (work in progress). Our clinical studies so far do suggest 3 out of 5 patients showing clinical responses and I believe we have reason to be encouraged.

Stewart (Ottawa): Do you have the histology of the two failures and did the primary tumor histology show tumor infiltrate? In a seminoma you might not have seen it.

Frenster (Stanford): We have reviewed all our histolgies and we don't notice a particular lymphocyte predominance in any of the patients pretreatment. I have not reviewed histology post-treatment so far.

Dent (McMaster): To my knowledge studies of in-vitro lymphocyte cytotoxicity have indicated a non-specific effect. The only study which shows specificity, is perhaps the one we heard this morning. Once you turn the lymphocyte on what guarantee do you have of specificity?

Frenster (Stanford): I recognize the similarity of your question to Dr. Falk's question, and the problem is the specificity of the therapeutic agent for the neoplastic versus the normal cells. We have of course the same problem with chemotherapeutic agents and we have reason to believe (Hellstrom I, et al, (1968), Nature 220: 1352) that a significant fraction of the lymphocytes in the blood of our tumour-bearing patients is sensitized for tumor antigens; therefore we hope that by non-specific stimulation this significant fraction would be stimulated for that particular tumour antigen. We have observed no significant limiting side effects such as autoimmune diseases which one might have anticipated if there were non-specific cytotoxicity being expressed in our patients.

Questioner: Do cells treated with PHA in this manner have a normal in-vivo half-life similar to untreated cells?

Frenster (Stanford): There is very little data about that. We are attempting to gather such data utilizing in-vitro labelled cells in that regard.

Questioner: Do you know how long your cells live in the body when reinjected?

Frenster (Stanford): No, nor whether there is recruitment of other lymphoid cells to the activated state, as one might hope.

ADDITIONAL REFERENCES

0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA. (Busch H, ed. 1974).

1. Archibald RB, and Frenster JH, "Quantitative Ultrastructural Analysis of In Vivo Lymphocyte- Reed-Sterberg Cell Interactions in Hodgkin's Disease", Natl. Cancer Inst. Monogr. 36: 239-245 (1973).

2. Frenster JH, and Rogoway WM, "In-Vitro Activation and Reinfusion of Autologous Human Lymphocytes", Lancet 2: 979-980 (Nov. 2, 1968).

3. Allen BL and Frenster JH, "Low-Dose Combination Chemotherapy of Disseminated Human Neoplasms". Lancet 2: 1324 (December 11, 1971).

4. Frenster JH, "Phytohemagglutinin-Activated Autochthonus Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N.Y. Acad. Sci. 277: 45-51 (1976).

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6. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites during Neoplastic Cell Differentiation within Hodgkin's Disease Lymph Nodes", in "Leukemia Review International", (Rich MA, ed.), Vol. 1, pp.22-23, Marcel Dekker, Inc., New York, 1983.

7.Keshgegian AA, Meisner LF, and Frenster JH, "Thymidine Reversal of Ribothymidine Inhibition of Lymphocyte Mitosis", in "Proc. 4th Ann. Leuk. Cult. Con. 1969, "(Mcintyre OR, ed.) pp. 361-366, (1971), New York, Appleton-Century-Crofts.

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