Killing of autologous B-lineage malignancy using CD3 x CD19 bispecific monoclonal antibody in end stage leukemia and lymphoma.

To develop an effective tumor immunotherapy for B-lineage non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), a bispecific monoclonal antibody (BsAb) has been generated with the first specificity for the CD3 epsilon-chain and the second for the CD19 antigen. Peripheral blood mononuclear cells (PBMCs) isolated from patients with NHL or ALL during remission or relapse rapidly proliferated (up to 179-fold increase) on in vitro activation combining phytohemagglutinin or CD3 monoclonal antibody with interleukin-2. After 3 weeks of stimulation, more than 90% of the PBMCs was CD3+ and CD8+, even when cultures were started with only 5% CD3+ cells. Cytotoxic activity against autologous malignant B cells was markedly enhanced (from 5% baseline to 70% lysis) by the addition of the CD3 x CD19 BsAb in all samples tested. Immunophenotypic examination of a series of tumor target cells showed that all samples examined showed CD54 (intercellular adhesion molecule-1) and HLA class I, but showed no B7 expression. CD11a (lymphocyte function-associated antigen-1) expression was heterogeneous. Various types of experiments showed that efficient CD3 x CD19 BsAb-mediated cytolytic capacity was not dependent on expression of either of these surface proteins. This contrasts with normal major histocompatibility complex-restricted antigen-specific cytotoxicity and may be essential for effective in vivo application of this BsAb.

Analysis of production, purification, and cytolytic potential of bi-specific antibodies reactive with ovarian-carcinoma-associated antigens and the T-cell antigen CD3.

OV-TL3 and MOv 18 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might therefore be used to develop therapies for ovarian cancer. The bi-specific MAbs alpha T3/OC2 and alpha OC/TR (both being combinations of MOv18 and alpha CD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOv18/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs alpha T3/OC2 and alpha OC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were highly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma supernatants was analyzed by immunohistochemistry, and by testing bi-specific MAb-mediated cytotoxicity against relevant target cells in the presence of effector cells. Stability testing of bi-specific MAb-producing hybridomas showed that, after sub-cloning, clones stably produced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with protein A and by ion-exchange high-pressure liquid chromatography, depending on the sub-class combination of the bi-specific MAb. The purified bi-specific MAbs were tested for their ability to mediate target-cell lysis with the use of cytotoxic T-cell clones and activated peripheral-blood lymphocytes. The purified alpha T3/OC2, alpha OC/TR, and OV-TL3/CD3 were all able to mediate highly specific lysis of various ovarian-carcinoma cell lines. No correlation was found between the level of antigen expression and bi-specific MAb-mediated cytolysis.

Production of bi-specific monoclonal antibodies in a hollow-fibre bioreactor.

In the present study we have measured the feasibility of producing high amounts of bi-specific monoclonal antibodies (MAb) for therapeutic purposes using a hollow-fibre bioreactor. We have studied the isotype composition and functional capacity of an OKT3/OV-TL 3 quadroma (IgG2a/IgG1) and we have compared the production of bi-isotypic MAb in this system with tissue culture flasks or mouse ascites. Both culture supernatant and purified bi-isotypic MAb were able to enhance the cytolytic activity of a CD8+ T cell clone against ovarian tumour cell lines, demonstrating the presence of functional bi-isotypic MAb (bi-specific MAb). The total amount of immunoglobulin produced after 38 days was 375 mg. After purification by protein A affinity chromatography, 79 mg of bi-isotypic MAb (IgG1/IgG2a) was obtained. This amount of bi-isotypic MAb was similar to the amount obtained from ascitic fluid produced by 200 mice or 38 l of tissue culture flask supernatant.

Optimization of culture conditions for activation and large-scale expansion of human T lymphocytes for bispecific antibody-directed cellular immunotherapy.

We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.

Enhancement of the lytic activity of cloned human CD8 tumour-infiltrating lymphocytes by bispecific monoclonal antibodies.

We have isolated tumour-infiltrating lymphocytes (TIL) clones from a patient with renal cell cancer. The cloning frequency and the effector function were measured. No difference in cloning frequency (r2 = 0.97, frequency = 1:13) was observed between TIL expanded with allogeneic versus autologous feeder cells. Sixty-four clones expanded with autologous feeder cells and 37 clones expanded with allogeneic feeder cells were assayed for cytolytic activity on an autologous primary renal cell carcinoma (RCC) culture, an allogeneic RCC line, and on the K562 and Daudi cell lines. Most of these clones were also phenotyped. Although TIL clones expressing cytotoxic activity for RCC lines could be generated with both feeder cell preparations, none of the clones tested showed specificity for cells from autologous primary RCC cultures. However, in the presence of relevant bispecific MoAbs (alpha OC/TR) all CD8+ TIL clones tested could be induced to lyse autologous RCC cultures. Furthermore, the cytolytic activity of all CD8+ clones tested against allogeneic RCC lines could be induced or further enhanced by alpha OC/TR or CD3/G250 bispecific MoAbs. In contrast, none of the CD4+ clones tested showed lytic activity. Quantitatively the cytotoxic response in the presence of alpha OC/TR or CD3/G250 of CD8+ TIL clones against G250+ and MOv18+ cell lines appears to be associated with the level of antigen expression on the target cells. Our results suggest that: (i) expansion of TIL with allogeneic or autologous feeder cells does not effect the lytic profile of the clones; (ii) the use of bispecific MoAbs may overcome a lack of specificity of cytotoxic T lymphocytes.

Bispecific antibodies reactive with the multidrug-resistance-related glycoprotein and CD3 induce lysis of multidrug-resistant tumor cells.

We describe the lysis of multidrug-resistant (MDR) tumor cells by various lymphocytic effector cells, retargeted with bispecific antibodies (heteroconjugates). The Ab-heteroconjugate used was prepared by chemically cross-linking the OKT3 monoclonal antibody (MAb) reactive with CD3 antigen on T lymphocytes, with the MRK16 MAb, which recognizes the MDR-associated P-glycoprotein. Cloned TCR alpha beta/CD3+ T lymphocytes, OKT3-activated peripheral-blood mononuclear cells and peripheral-blood mononuclear blood lymphocytes, stimulated with allogeneic irradiated cells in a mixed lymphocyte culture, could be induced to lyse MDR ovarian tumor cells in the presence of Ab-heteroconjugate CD3/MRK16, whereas the drug-sensitive parental tumor cells lacking the P-glycoprotein were not lysed by these retargeted effector cells. Cloned TCR gamma delta/CD3+ T lymphocytes showed a high MHC-unrestricted lysis of MDR tumor cells. Addition of Ab-heteroconjugate CD3/MRK16 could therefore not enhance target-cell lysis. Melanoma tumor cells transfected with the mdr-I gene which codes for the P-glycoprotein were also efficiently lysed by Ab-heteroconjugate retargeted cloned TCR alpha beta/CD3+ T cells. Tumor cell lines derived from organs known to express the P-glycoprotein also were lysable by the retargeted effector cells.

Coordinated V gamma and V delta gene segment rearrangements in human T cell receptor gamma/delta+ lymphocytes.

Monoclonal antibodies (mAb) were used to characterize a panel (n = 46) of T cell receptor (TcR) gamma/delta+ T cell clones. Three of these antibodies have been described to react with specific variable region-encoded protein products and can therefore be used to detect functional gene rearrangements. The majority of peripheral blood-derived clones (43 out of 45) expressed the epitopes recognized by mAb BB3, encoded by the V delta 2 gene segment and mAb Ti gamma A, encoded by the V gamma 9 gene segment. These clones lacked the antigenic determinant recognized by mAb delta-TCS-1, encoded by the V delta 1 gene segment. The other two peripheral blood-derived clones and an ascites-derived clone were Ti gamma A-, BB3- and delta-TCS-1+. Biochemical analysis revealed that all Ti gamma A+, BB3+ T cell clones expressed the disulfide-linked form of the receptor. The two peripheral blood-derived delta-TCS-1+ T cell clones expressed the nondisulfide-linked form whereas the ascites-derived delta-TCS-1+ clone, AK119 expressed the disulfide-linked form of the TcR gamma/delta heterodimer. This indicates that V delta 1-encoded delta chains can be associated either with a C gamma 1- or a C gamma 2-encoded gamma chain. The preferential use of certain V gamma and V delta gene segments suggests the existence of a limited combinatorial diversity in TcR gamma/delta heterodimers, i.e. Ti gamma A+ (V gamma 9), BB3+ (V delta 2) and delta-TCS-1- disulfide-linked heterodimers and Ti gamma A-, BB3- and delta-TCS-1+ (V delta 1) disulfide- or non disulfide-linked forms.

Induction of lysis by T cell receptor gamma delta+/CD3+ T lymphocytes via CD2 requires triggering via the T11.1 epitope only.

The requirements for activation of the lytic machinery through CD2 of TCR gamma delta+/CD3+ cells were examined, by utilizing bispecific heteroconjugates containing anti-CD2 mAb cross-linked to anti-DNP. Contrary to the CD2 activation requirements in TCR alpha beta+/CD3+ cells, cytotoxic activity in TCR gamma delta+/CD3+ clones and TCR-/CD3- NK cell clones can be induced by heteroconjugates containing a single anti-CD2 (OKT11.1) mAb. Activation of TCR gamma delta+/CD3+ cells via CD2 is independent of heteroconjugates binding to CD16 (Fc gamma RIII), because heteroconjugates prepared from Fab fragments induced equal levels of lysis. Moreover, anti-CD16 mAb did not inhibit triggering via CD2 in TCR gamma delta+/CD3+ cells. In TCR-/CD3- NK cells, however, induction of cytotoxicity via CD2 is co-dependent on interplay with CD16. Anti-CD3 mAb blocked the anti-CD2 x anti-DNP heteroconjugate-induced cytotoxicity of TCR gamma delta+/CD3+ cells, indicating a functional linkage between CD2 and CD3 on these cells. We conclude that induction of lysis via CD2 shows qualitatively different activation requirements in TCR gamma delta+/CD3+, TCR alpha beta+/CD3+ CTL and TCR-/CD3- NK cells.

Leucoagglutinin induces differential proliferation of lymphocyte subsets.

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.

Distinct molecular forms of human T cell receptor gamma/delta detected on viable T cells by a monoclonal antibody.

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells.

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.

Two types of gamma T cell receptors expressed by T cell acute lymphoblastic leukemias.

CD3+ cells, isolated from peripheral blood of two patients with T cell acute lymphoblastic leukemia (T-ALL), did not react with the monoclonal antibody WT31, which is thought to recognize a framework determinant on the conventional T cell receptor (TcR), consisting of disulfide-linked alpha and beta chains. The T-ALL cells of neither patient synthesized TcR alpha mRNA; the cells of patient DD contained only truncated (D-J) TcR beta mRNA, while the cells of patient HZ contained truncated as well as mature (V-D-J) TcR beta mRNA. The leukemic cells of both patients made TcR gamma mRNA. At the cell surface, the T-ALL cells of patient DD expressed a CD3-associated disulfide-linked dimer, which contained the TcR gamma protein. On the leukemic cells of patient HZ the TcR gamma protein was present as a 41-44-kDa CD3-associated subunit in a noncovalently linked form. The TcR gamma genes in the T-ALL cells of patient DD were rearranged exclusively to the C gamma 1 locus, while in the T-ALL cells of patient HZ both C gamma 1 alleles were deleted and rearrangement to the C gamma 2 locus had occurred. The C gamma 1 gene segment, just like the TcR alpha and TcR beta gene segments, contains a cysteine codon in its second exon. This cysteine residue is involved in the formation of the interchain disulfide bond. The human C gamma 2 gene segment, however, does not contain a cysteine codon in its second exon. The absence of the cysteine residue in C gamma 2 encoded TcR gamma chains explains the lack of an interchain disulfide bond in the TcR on the T-ALL cells of patient HZ. The TcR gene configuration, as well as the expression of model for T cell differentiation in which the TcR gamma gene rearranges first to the C gamma 1 locus prior to or coinciding with D-J joining of the TcR beta gene, followed by rearrangement to the C gamma 2 locus and V-D-J joining of the TcR beta gene.

Regulation of cytolytic activity in CD3- and CD3+ killer cell clones by monoclonal antibodies (anti-CD16, anti-CD2, anti-CD3) depends on subclass specificity of target cell IgG-FcR.

Anti-CD3 MAb can inhibit MHC-restricted cytolytic activity of CD3+ mature cytotoxic T cells. In particular effector-target cell combinations, however, anti-CD3 MAb enhance or induce cytolysis by cross-linking CD3+ effector and IgG-FcR+ target cells. Virtually all natural killer (NK) cells or NK cell-derived clones are CD3-4-8- but do express CD2 and CD16 (IgG-FcR) antigens. We have studied how these cell surface molecules are involved in the regulation of cytolytic activities. The addition of anti-CD2 MAb to effector and target cells was found to induce conjugate formation of the IgG-FcR+ target cells with the effector cell and nonspecific cytolysis of, for instance, the P815 mouse mastocytoma cells. Enhancement or induction of conjugate formation and cytolysis of IgG-FcR+, P815, U937, and Daudi cells was also accomplished by using anti-CD16 MAb (e.g., Leu-11c (B73.1) or CLB Fc-gran 1 (VD2) MAb). Some human and mouse tumor cell lines (K562, P815, and U937) appear to express distinct types of IgG-FcR, showing different affinities for distinct subclasses of MAb (e.g., IgG1, IgG2a), but another line (Daudi) expresses only one type of IgG-FcR preferentially binding IgG1 MAb. Here we demonstrate that IgG-FcR on the effector cells can act as activation sites because anti-CD3 as well as anti-CD16 MAb of IgG1 and IgG2a subclasses can induce lytic activity of target cells bearing the relevant IgG-FcR. These data demonstrate that induction of conjugate formation and cytolysis by MAb occur when the target cells bear IgG-FcR with "specificity" for those MAb. Thus, besides via CD3, cytolytic activity by mature T and NK cells also can be induced via the CD2 and CD16 antigens on these cells.

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1.

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.

A T-cell receptor gamma/CD3 complex found on cloned functional lymphocytes.

Cloned blood lymphocytes that do not express the alpha- and beta-chains of the T-cell receptor show MHC-unrestricted cytotoxicity. These cells carry the gamma-protein, disulphide-linked either to another molecule or to itself, and associated with the CD3 complex. These observations may help to solve the mystery posed by the discovery of the gamma gene.

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes.

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.