Bone marrow transplantation across major histocompatibility barriers. V. Protection of mice from lethal graft-vs.-host disease by pretreatment of donor cells with monoclonal anti-Thy-1.2 coupled to the toxin ricin.

A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and thus prevent graft-vs.-host disease (GVHD). The method utilizes a monoclonal antibody directed at the Thy-1.2 antigen but is complement independent. To make anti-Thy-1.2 toxic, the antibody is covalently linked to the toxin ricin. Ricin ordinarily binds, enters, and kills cells through receptors containing galactose. The hybrid protein, anti-Thy-1.2-ricin, can enter and kill cells via the Thy-1.2 receptor. In the presence of lactose the usual entry route for ricin is largely blocked and the hybrid is shown to be a highly selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes. We have used a model of severe and fatal GVHD where BALB/c splenocytes and bone marrow cells are given to irradiated C57BL/6 recipients. Over 90% of these mice die by day 70, exhibiting signs of GVHD. When donor cells are pretreated with 0.5 microgram/ml of anti-Thy-1.2-ricin plus 200 mM lactose before injection, 10 of 11 animals survive through day 70 without signs of GVHD. These studies demonstrate that ricin linked to monoclonal antibodies may have utility related to the prevention of GVHD in human bone marrow transplantation.

P-24: a human leukemia-associated and lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody.

This study was directed at surface structures that are found on human lymphohemopoietic progenitor cells in normal and leukemic bone marrow. A monoclonal antibody was produced against an acute lymphoblastic leukemia (ALL) cell line of the pre-B phenotype; this antibody (BA-2) was used to demonstrate a cell surface polypeptide of approximately 24,000 mol wt that migrates similarly in both reduced and nonreduced form. This polypeptide, p24/BA-2, was shown by immune precipitation and gel electrophoresis and cell distribution studies to be different from HLA-DR and gp 100/cALLa. p24/BA-2 was present on the surface of 77% (54/70) of cases of non-T, non-B ALL; BA-2 staining was less bright or nondetectable in surface Ig+ (SIg+) chronic lymphocytic leukemia (CLL) and T ALL and nondetectable on peripheral T and B lymphocytes. Approximately 3% of bone marrow mononuclear cells were p24/BA-2+, and these cells were E rosette-, surface (SIg-), and nonphagocytic. Marrow TdT+ progenitor cells were frequently p 24/BA-2+. Results suggest that p24/BA-2 represents a surface structure present on lymphohemopoietic bone marrow progenitor cells and that most common types of ALL bear the p25/BA-2 structure.

Use of a novel colony assay to evaluate the cytotoxicity of an immunotoxin containing pokeweed antiviral protein against blast progenitor cells freshly obtained from patients with common B-lineage acute lymphoblastic leukemia.

We report a novel colony assay for B-lineage progenitor cells in acute lymphoblastic leukemia (ALL). The primary plating efficiency of blast progenitors freshly obtained from common B-lineage ALL patients varied between 0.09 and 2.63%. Morphological, cytochemical, and immunological analyses of cells from day 7 colonies provided the evidence that they are B-lineage lymphoblasts. Immunological marker analyses of cultured blasts using BA-2 (anti-CD9), BA-3 (anti-CD10), BA-1 (anti-CD24), and B43 mAb have allowed us to define two distinct immunological groups. The first group had BA-2+, BA-3+, BA-1+, B43+ marker profiles, consistent with the phenotype of uncultured bone marrow blasts. The second group differed in that the cells in the blast colonies were BA-3 (anti-CD10)-negative, although many of the cells in the bulk population were BA-3+ before culture. Blasts from both groups were able to proliferate and form secondary colonies when recultured. A pan-B immunotoxin was synthesized by linking B43, a human B cell-specific mAb, to pokeweed antiviral protein (PAP). This study showed that B43-PAP can effectively eradicate leukemic progenitor cells freshly obtained from patients with common B-lineage ALL. B43-PAP eliminated greater than 99.96% of blast progenitors under conditions in which only minimal inhibition of normal bone marrow progenitor cells (CFU-GM, CFU-E, CFU-MK, CFU-GEMM) was observed. Our results establish that the surface determinant recognized by B43 is expressed on B-lineage progenitor cells in ALL, and that these cells are sensitive to PAP at the ribosomal level. To our knowledge, B43-PAP is the first IT to prove effective against common B-lineage ALL cells.

Mature T-lineage leukemia with growth factor-induced multilineage differentiation.

We report an acute T-lymphoblastic leukemia with a predominantly mature CD3+ CD7+ WT31+ phenotype that was induced to differentiate into different cell lineages by various recombinant human growth factors. In the presence of IL-3 or GM-CSF, the leukemic cells gave rise to myeloid and monocytic cells including terminally differentiated, partially functional, segmented neutrophilic granulocytes as assessed by morphologic, cytochemical, immunophenotypic, and functional criteria. In the presence of IL-2, leukemic granulated lymphoid cells exhibiting MHC-unrestricted cytotoxicity and expressing a CD2+ CD3+ CD5+ CD7+ CD8+ CD33+ WT31+ Leu19+ phenotype arose. Leukemic cell cultures initiated with IL-3 yielded growth factor-independent cells with a mixed lineage phenotype and morphologic and cytochemical evidence of immature blasts. These were T lymphocyte and myeloid surface antigen (CD2,CD3,CD5,CD7,CD13,CD33,WT31) positive. Identical rearrangements of the constant region of the TCR-delta gene and of the joining regions of the TCR-beta, -gamma, and -delta genes were observed in the fresh and all cultured leukemic cells, indicating that they were derived from the same malignant clone. Consistent with the molecular genetic data, the cytogenetic analyses of the GM-CSF-, IL-3-cultured and the growth factor-independent leukemic cells showed the presence of multiple, closely related abnormal clones, all of which had an interstitial deletion of part of the long arm of chromosome 6 and a complex 1;10;12 translocation. In conclusion, these data demonstrate the involvement of a multipotent leukemic precursor cell in this predominantly mature CD2+ CD3+ CD5+ CD7+ WT31+ T-ALL. This multipotent leukemic precursor may be susceptible to various growth factors and respond with ordered differentiation and maturation.

Acute lymphoblastic leukemic cells with T (thymus-derived) lymphocyte markers.

Five of nine children with acute lymphoblastic leukemia had lymphoblasts that bound sheep erythrocytes or reacted with antiserum to thymocytes, suggesting involvement of T (thymus-derived) cells. When lymphoblasts from all patients were examined by immunofluorescence they were found to lack a marker for B (bone marrow or bursa-equivalent) cells, that is, the presence of surface immunoglobulins.

Anti-T-cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies.

Three new reagents that react against human T cells were synthesized by covalently linking the toxin ricin to monoclonal antibodies recognizing differentiation antigens on the surface of T lymphocytes. Each of these immunotoxins selectively inhibited T-cell proliferation when the cells were incubated in the presence of lactose. Multipotent human stem cells were inhibited only at much higher concentrations. Mixtures of all three immunotoxins were more effective than any one alone. These reagents have the potential for preventing graft-versus-host disease in man.

T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region.

Heterogeneity of cultured leukemic lymphoid progenitor cells from B cell precursor acute lymphoblastic leukemia (ALL) patients.

Colony assays were performed for 50 patients with B cell precursor acute lymphoblastic leukemia (ALL). Blast colony formation was observed for 33 patients, and the plating efficiency (PE) showed a marked interpatient variation, which indicates a pronounced biological heterogeneity at the level of leukemic progenitor cells. Notably, the mean PE of leukemic B cell precursors from patients with a pseudodiploid or near-diploid karyotype with structural chromosomal abnormalities (SCA) was significantly higher than the mean PE of normal diploid or hyperdiploid cases. All patients who had SCA involving 7p13, 11q23-24, or 12p11-13, and patients with a Philadelphia chromosome had high PE values. The S phase percentage, expression of CD19 antigen, and relapse status were also correlated with PE. Significantly, colony blasts had slightly different surface marker profiles in each case and were common ALL antigen negative in 33% of cases, which indicates the existence of a marked immunological heterogeneity at the level of leukemic progenitor cells.

Acute leukemia expressing the gamma gene product of the putative second T cell receptor.

Early thymus-derived lymphocytes bearing the T gamma gene product in association with the CD3(T3) complex have recently been described. We report a unique case of human acute lymphoblastic leukemia with a CD2+, CD3+, CD4-, CD5+, CD7+, CD8-, WT31- phenotype. These cells were found to have T gamma gene rearrangement and T gamma transcripts in absence of T alpha or T beta rearrangement or transcripts. Immunoprecipitation studies with anti-CD3 antibodies showed a 43-kD protein associated with T3; this 43-kD protein is also precipitated with antiserum raised against synthetic peptides representing the constant region of the putative T gamma protein.

Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes.

Cytogenetic studies have previously identified abnormalities of chromosome band 11q23 in many cases of infant acute leukemia. Recent studies by ourselves and others have demonstrated breakpoint clustering in acute leukemias bearing translocations involving 11q23, and a Drosophila trithorax gene homologue (called MLL, HRX, or ALL-1) has been shown to span the 11q23 breakpoints of these translocations. To determine if this gene is affected in infant acute myeloid leukemia (AML), we have analyzed 26 infant AML cases for molecular alterations of this 11q23 gene. 15 out of 26 cases studied (58%) showed rearrangement of the MLL gene at the molecular level, and these rearrangements were clustered within an approximately 11-kb region containing nine exons of this gene. Moreover, 14 of the 15 cases with 11q23 rearrangements (93%) had myelomonocytic or monocytic phenotypes (M4 or M5 FAB subtypes, respectively), both of which are associated with a poor prognosis in childhood AML. In contrast, only 1 of 11 nonrearranged cases had an M4 or M5 phenotype (P = 0.00002). Rearrangement also correlated significantly with hyperleukocytosis (P = 0.02), another clinical parameter associated with poor outcome in this disease. Our results demonstrate that molecular rearrangements of MLL are common in M4 or M5 infant AML, and suggest that alteration of this gene may result in abnormal control of proliferation and differentiation in monocytic progenitor cells.

Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins.

We have explored the relationship among immunoglobulin gene rearrangement, cytoplasmic immunoglobulin production, and cell surface antigen expression within 37 cases of acute lymphocytic leukemia. All 12 cases of the T cell type had germ-line kappa and lambda genes and 11 of 12 had germ-line heavy chain genes. In contrast, all 25 cases of the "non-T, non-B" classification, which lacked both definitive T cell markers and surface immunoglobulin, had rearranged immunoglobulin genes, indicating that they represent precursor cells already committed to the B cell lineage at the gene level. 14 had rearranged heavy chain genes, yet retained germ-line light chain genes, whereas 11 cases had both heavy and light chain gene reorganizations. All patterns of immunoglobulin gene rearrangement predicted by a model that proceeds from heavy chain gene recombination to light chain genes were observed. Despite the uniform presence of rearranged immunoglobulin genes, only five cases produced cytoplasmic mu-chain, one exceptional case produced gamma-chain, and another produced only lambda-chain. The cases of B cell precursor type that do not produce immunoglobulin may represent cells that frequently possess ineffectively rearranged immunoglobulin genes. Included in this group may be a set of cells trapped within the B cell precursor series because their ineffective rearrangements have eliminated certain gene subsegments necessary for the assemblage of an effective heavy chain gene. All seven cases of the non-T, non-B subgroup that bore HLA-DR but lacked CALLA (the common acute lymphocytic leukemia-associated antigen) represented the earliest recognizable stage of B cell precursors with rearranged heavy chain genes but germ-line light chain genes. Correlations here suggest that cells entering B cell development express HLA-DR and rearrange heavy chain genes before the expression of a B cell-associated antigen recognized by the antibody BA-1, the antigen CALLA, and any subsequent light chain gene rearrangements.

Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway.

17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90, results in cell type-specific inhibition of proliferation of leukemic cells. GTP14564 is a tyrosine kinase inhibitor actively against FLT3. The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the role of FLT3 in their inhibitory effects. The importance of FLT3 mutations was demonstrated using small interfering RNA (siRNA) targeted to FLT3. Similar to FLT3 siRNA, GTP14564 inhibited FLT3 internal tandem duplication (ITD) cells (MV4;11) and FLT3 amplified wild-type cells (SEMK2-M1), but not wild-type FLT3 cells (RS4;11). However, when RS4;11 cells were stimulated with FLT3-ligand, phosphorylation of STAT5 and GTP14564 inhibition were observed. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells. We observed synergistic effects of combined 17-AAG and GTP14564 in cell lines with FLT3-ITD and amplified wild-type FLT3. Combined treatment with 17-AAG and GTP14564 reduced the levels of p-FLT3 and p-STAT5, enhanced G0/G1 arrest and apoptosis in FLT3-ITD and amplified wild-type FLT3. The combination of 17-AAG with FLT3 kinase inhibitors can enhance targeted therapy in leukemias with FLT3 mutations, such as MLL fusion gene leukemias.

Antigens associated with acute leukemia detected in the primed lymphocyte test.

Mixed lymphocyte culture (MLC) studies of families with several leukemia patients, all potential bone marrow transplant recipients, demonstrated that cells from acute myelogenous leukemia patients (5 of 5) and acute undifferentiated leukemia patients (1 of 4) in relapse stimulated autologous lymphocytes as well as lymphocytes from siblings known to be identical at the major histocompatibility linkage group. In the patients studied, the blast transformation induced by leukemia cells was not detectable when the patient was in remission. Stimulation by leukemia cells also elicited increased responses of the lymphocytes from normal haploidentical siblings, parents, and unrelated individuals as compared to stimulation by normal allogeneic cells or leukemia cells of patients with leukemia in remission. The primed lymphocyte test (PLT) was used successfully to establish HLA-D identity of the leukemia patients and their respective HLA-identical siblings, despite high percentages of circulatory blasts. Utilizing lymphocytes from normal siblings primed against the leukocytes from an HLA-identical sibling with leukemia, we also presented results of PLT's which suggested that the stimulation induced by leukemia cells in MLC was produced by leukemia-associated antigens.

Immunologic and ultrastructural heterogeneity of acute lymphoblastic leukemia-associated antigen-positive human leukemias.

To further examine the heterogeneity of human acute lymphoblastic leukemia, an immunologic and ultrastructural study was undertaken on 14 patients positive for the acute lymphoblastic leukemia-associated antigen (ALLA). In malignant cells from 7 of the patients studied, cytoplasmic granular inclusions that resembled those seen in leukemia mast cells and basophils (M-B granules) were evident. Four patients had malignant cells expressing the pre-B-cell phenotype (i.e., cytoplasmic IgM-positive and surface immunoglobulin-negative), and 3 patients had malignant cells that lacked both M-B granules and cytoplasmic IgM. These results support the existence of distinct phenotypic subgroups within the ALLA+ leukemias.

Establishment of a human t(4;11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin.

Human acute leukemia, with a chromosomal translocation involving chromosomes 4 and 11, t(4;11)(q21;q23), is the most common form of leukemia in infants and responds very poorly to conventional therapy. A human CD19+ mixed-lineage leukemia cell line with a t(4;11)(q21;q23) translocation, RS4;11, disseminated and proliferated in the hematopoietic tissues and other organs of mice with severe combined immunodeficiency in a manner similar to that observed in humans and killed 100% of the animals. The anti-CD19(B43)-pokeweed antiviral protein immunotoxin selectively inhibited clonogenic RS4;11 cells in vitro, markedly reduced the burden of disseminated leukemia of severe combined immunodeficient mice, and, most importantly, resulted in the long-term survival of treated animals. This severe combined immunodeficient mouse model should be useful for the design of more effective treatment strategies for refractory human leukemias.

Heterogeneity in MLL/AF-4 fusion messenger RNA detected by the polymerase chain reaction in t(4;11) acute leukemia.

We have designed a single polymerase chain reaction (PCR) primer pair that detects the MLL/AF-4 fusion mRNA encoded by the derivative 11 chromosome from t(4;11)(q21;q23) leukemia cells using the reverse transcriptase PCR technique. PCR amplification was possible in seven of seven cells studied. Sequencing of the amplified products showed three different breakpoints on 11q23 and three on 4q21, resulting in six unique fusion sequences. All fusion sequences maintained an open reading frame. The areas of the MLL and AF-4 genes that are conserved in all derivative 11 fusion RNAs and therefore likely to contribute to the function of the oncogenic fusion protein are centromeric regions of MLL through exon 6 (retaining the AT hook motif) and telomeric regions of AF-4 beginning at codon 491 (containing nuclear localization and GTP-binding motifs). A single primer pair was able to detect the derivative 11 fusion transcript in seven of seven cases of t(4;11) acute leukemia tested. Given the variability shown in specific fusion sequences, studies correlating differential exon usage with clinical parameters will require different fusion-specific oligonucleotides or PCR primer pairs.

Successful treatment of human acute T-cell leukemia in SCID mice using the anti-CD7-deglycosylated ricin A-chain immunotoxin DA7.

The study of new therapeutic approaches for refractory human leukemia has been hampered by the lack of relevant in vivo models with disseminated disease, particularly T acute lymphoblastic leukemia (T-ALL). In the present study we evaluated methods for establishing and therapy of a human T-ALL cell line (MT-ALL) in 73 SCID mice. MT-ALL is a T-cell receptor alpha/beta +, CD3+, and CD7+ leukemia cell line, derived from a patient with refractory disease and early death. Injection of 5 x 10(7) MT-ALL cells i.v. caused disseminated human leukemia in hematopoietic and nonhematopoietic organs in 100% of SCID mice (n = 9) leading to death or terminal disease at 65 to 70 days after a uniform clinical course. To study possible therapeutic approaches for disseminated leukemia we utilized an immunotoxin, DA7, constructed by chemically linking the mouse IgG2b anti-CD7(3A1E) monoclonal antibody which recognizes a pan-T-cell marker expressed on almost all T-cell leukemias to deglycosylated ricin A-chain, a catalytic plant toxin and inhibitor of protein synthesis. Administration of DA7 led to greater than 5 log kill of clonogenic MT-ALL cells in vitro and selectively inhibited protein synthesis. DA7 was administered to mice at a dose of 10 micrograms/mouse/day for 5 consecutive days starting 8 days after i.v. inoculation of leukemia. The immunotoxin therapy resulted in significant long term survival over 348 days compared to untreated or control mice treated with anti-CD7 antibody and deglycosylated ricin A-chain which were all dead by day 70 (P less than 0.001). Even after more than 11 months there was no evidence of disease in 82% of the DA7 treated animals. SCID mice given i.p. injections (n = 9) developed an i.p. tumor mass but demonstrated metastasis outside the peritoneum with disseminated leukemia in hematopoietic and nonhematopoietic organs, a finding different from most conventional nude mouse models. The leukemia was fatal in 100% and killed the animals at 68-95 days. SCID mice given i.p. injections of MT-ALL completely responded to therapy with DA7, resulting in survival of 100% of the animals (n = 10) at 216 days (P less than 0.001 compared to untreated animals). Anti-CD7 antibody, deglycosylated ricin A-chain, and a control anti-melanoma immunotoxin (IND1-RTA) showed no therapeutic effect. We conclude that DA7 is an effective in vivo therapeutic agent against human MT-ALL in the SCID mouse system, suggesting potential usefulness for therapy of humans with poor prognosis T-cell leukemia.

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen.

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.

Use of monoclonal antibodies, morphology, and cytochemistry to probe the cellular heterogeneity of acute leukemia and lymphoma.

A combined immunological, morphological, and cytochemical approach to the study of malignant cells in patients with acute leukemia and lymphoma is presented. Newly produced monoclonal antibodies that bind to antigens of human mononuclear cells (TA-1), or B-lymphocytes (BA-1) were used to study malignant cells from patients with acute lymphoblastic leukemia (ALL). acute myelocytic leukemia, acute myelomonocytic leukemia, and chronic lymphocytic leukemia. Results in lymphoid leukemia-lymphoma patients were compared with other immunological markers and indicate that the major groups of ALL and childhood non-Hodgkin's lymphoma are T-ALL, pre-T-ALL, pre-B-ALL, B-ALL, and non-T, non-B-ALL. In addition, each major group had multiple phenotypes when analyzed with seven immunological markers including the erythrocyte rosette receptor, surface immunoglobulin, cytoplasmic immunoglobulin M, the early lymphocyte-acute lymphoblastic leukemia antigen, monoclonal antibody TA-1, monoclonal antibody BA-1, and a monoclonal antibody against HLA-DR. While immunological heterogeneity was demonstrable within each group, distinct biological behavior was observed, with T-ALL and B-ALL generally presenting as "lymphomas" and the others presenting as "leukemias." Morphological analysis using the French-American-British classification provided independent information in the definition of groups with differing clinical behavior. Cytochemical analyses demonstrated focal paranuclear staining of leukemia cells with acid phosphatase in 73% of T-ALLs and 6% of non-T, non-B-ALLs.

Biochemical characterisation of leukaemia-associated antigen p24 defined by the monoclonal antibody BA-2.

The leukaemia-associated cell surface antigen p24/BA-2 is a single polypeptide chain with a molecular weight of 24,000. Treatment with glycosidases or exposure of cells to tunicamycin failed to show any change in the molecular weight of the antigen when examined by SDS-polyacrylamide gel electrophoresis. In addition, it failed to bind to lectin affinity columns of concanavalin A, lentil lectin or ricinus communis lectin. This is consistent with the absence of N-asparagine linked oligosaccharide chains on the antigen. Pulse-chase labelling of protein p24 shows a post-translational modification resulting in a molecular weight increase of approx. 500-1000. Alkaline treatment resulted in a decrease in molecular weight of approximately the same amount, suggesting that p24 contain some O-glycosidically linked oligosaccharide. Protein p24 has a basic pI of 7.3 which is unchanged after neuraminidase treatment. Protein P24/Ba-2 cannot be labelled by either the lipophilic photoactivatable nitrene reagent, hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine, or with [32P]phosphate. This suggests that the molecule is non-integral in nature and that it does not form an intimate association with the lipid matrix. Identical molecular weights, when reduced and non-reduced antigens were compared, suggest that it contains no internal disulphide linkages and failure to detect any other band on gradient gel SDS-polyacrylamide gel electrophoresis from 5-15% suggests that is is not strongly associated with any other structure.