new advances in cellular therapy · purpose. these include ligands, peptides, chimeric ligands,...

New Advances in Cellular Therapy

Mehrdad Abedi MDAssociate Professor of Medicine

17th Annual Advances in OncologySeptember 30-October 1, 2016

Sacramento, CA

Mehrdad Abedi, M.D.Update on Cellular Therapies for Hematologic Malignancies.

Relevant financial relationships in the past twelve months by presenter or spouse/partner:

Grant/Research Support: CIRM, Kite Pharmaceutical, Celgene, AmgenConsultant/Speaker Programs: Celgene, Millenium, BMS, Gilead

The speaker will directly disclosure the use of products for which are not labeled (e.g., off label use) or if the product is still investigational.

Type of cellular therapy

T cell therapyNK Cell therapy

Stem cell therapyDendritic cell therapy

Mesenchymal Cell Therapy

• Bispecific antibodies

• BsAbs are reagents that combine the specificities of two antibodies in a single molecule.

• Using quadroma technology, two different hybridoma cells are fused to raise a cell that can simultaneously produce the two parental heavy (H) and light (L) chains that join spontaneously by Fc pairing forming

heterodimers• It should also be pointed out that the effector activity of

nonlymphoid cells and natural killer (NK) cells has been exploited in retargeting approaches

At least 40 different ways to generate BsAbs have been described

(A) IgG-based BsAbs

(B) FAb-based BsAbs.

(C) Fv-based BsAbs

(D) scFv-based BsAbs with a protein spacer into the linker

(E) Variants of scFv–Fc

Adaptive Cell Transfer Therapy

Adoptive cell therapy (ACT) is a treatment that uses a cancer patient’s own T lymphocytes with anti-tumour activity, expanded in vitro and reinfused into the patient with cancer.

Adaptive Cell Transfer Therapy

• TIL( Tumor infiltration T-lymphocytes therapy)

• TCR ( T-cell receptor therapy)

• CAR-T (Chimeric antigen receptor T-cell therapy)

Rosenberg, S. A.et al.Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. Preliminary report. N. Engl. J. Med. 319, 1676–1680 (1988).

TIL

3. Infuse the "boosted" T-cells into the patient.

Overview: Adoptive T cell therapy

1. Isolation of TILs or tumor specific T-cells from blood

2. Expand and activate T-cells ex vivo

Target therapy with Tumor specific T cells

Cancer: Melanoma

Autologous tumor infiltrating lymphocytes (TILs); “Live drug”

AdvantagesHigh response rate (>50%),

Long-term remission,

Less toxic & gentler to the patient

Limitation: Extraction of TILs,

Cell manufacturing

Possible alternateT cell Engineering (CAR-T cells)

Rosenberg SA & Dudley ME 2009 Current Opinion of Immunology

Morgan, R. A.et al.Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129 (2006).

TCR

ITAM: immunoreceptor tyrosine-based activation motifTCR complex :TCR, CD3, ζ

TCR

• The Antigen targeting domain• The spacer/hinge domain• The transmembrane domain• The signalling domain

Antigen targeting by CAR molecules most commonly involves the use of scFv. However, several alternative targeting moieties may also serve this purpose. These include ligands, peptides, chimeric ligands, receptor derivatives and single domain antibodies

some reports have suggested that different hinge regions might critically control surface expression levels, construct stability and antigen binding affinity, which directly influence the efficiency of CAR-redirected effector functions

the targeting and signaling properties of CARs, focusing on their effects on T-cell specificity, potency, and safety.

CARs and TCRs have their respective advantages and

disadvantages

• Although the flexibility and “dynamic range” of CARs is attractive, current CARs are limited to recognizing cell surface antigens

• CARs, however, do not require antigen processing and presentation by HLA and are therefore more broadly applicable to HLA-diverse patient populations

• In this regard, CARs provide a broader range of functional effects than transduced T-cell receptors (TCR), wherein strength of signaling, which is for the most part determined by the TCR’s affinity for antigen, is the principal determinant of T-cell fate.

• CARs ﹠TCRs more secure , better targeted , more persistent

3rd generation CARs

Wang et alHum Gen Ther 2007

Zhao et al JI 2009

Wilkie et alJI 2008

Adoptive T cell therapy: CAR-T cells

Antigen specific domain

CAR-T cells (Chimeric antigen receptor-T cells0

T cells transduced with tumor-specific CAR

CAR: Single fusion molecule with antigen specificity plus signaling domain

Three types of CAR: First/second/generations

Based on co-stimulatory receptors

Cancer: Solid tumor & hematological malignancies

Maus M V et al. Blood 2014;123:2625-2635

“Live drug”

Tumor recognition independent of HLA

(no HLA typing needed)

Multiple anti-tumor immuno-modulators can be engineered

Target variety of antigens (protein,

carbohydrate, glycolipid)

Advantages of CAR T cells

What is the ideal TAA?

• Qualities of the “ideal” tumor antigen:

– Expression restricted to the tumor cell population alone

– Restricted expression to tumor and otherwise non-vital tissues

– Expressed by all tumor cells– Expressed on the tumor cell surface– The target antigen is required by the tumor

cell for survival

CAR-targeted TAAs in clinical trials

Jena et al Blood 2010

Clinical significance of CAR-T cells

Target CAR Cancer Objective response

CD19 CAR:CD28-CD3ζ Lymphoma and CLL

N=7: 1CR, 5 PR & 1SD

CAR:CD137-CD3ζ ALL 2CR

CAR:CD28-CD3ζ ALL 5CR

CD20 CAR:CD137-CD28-CD3ζ

NHL N=3: 1PR, 2NED

CEA CAR-CD3ζ (1st gen) Colorectal & breast

N=7: minor responses in two patients

GD2 CAR-CD3ζ (1st gen) Neuroblastoma N=19: 3CR

ERBB2 CAR:CD28-CD137-CD3ζ

Colorectalcancer

N=1, patient died

Kershaw et. al. 2013 Nature Reviews cancer

Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. Aug 25 2011;365:725-33

Expression of CD19 and other B cell markers on B lineage

cellspreB-ALL

B cell lymphomas and leukemias myelomas

Stem Cell pre B immature B mature B plasma cellpro B

CD19

CD22

CD20

Figure 1 Clinical Response in the Patient.

Figure 1 Clinical Response in the Patient.

Before treatment 6 months after treatment

Response in Patient with Refractory DLBCL

Depth of Best Response in NCI B Cell Lymphoma Study

6confidential

Figure 2 Serum and Bone Marrow Cytokines before and after Chimeric Antigen Receptor T-Cell Infusion.

Figure 3.Expansion and Persistence of Chimeric Antigen Receptor T Cells In Vivo.

Failure• Morgan RA, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR

gene therapy. J Immunother 2013;36:133–151.

• Morgan RA, Yang JC, Kitano M, Dudley ME,Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol Ther 2010;18:843–851.

• Parkhurst MR, et al. T cells targeting carcinoembryonic antigen can mediateregression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011;19:620–626.

• Brentjens R, Yeh R, Bernal Y, Riviere I, SadelainM. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther 2010;18:666–668.

Challenges of CAR-T

Target selection

Optimize costimulatory signaling of T cell effector functions

Toxicities (on-target but off-tumor toxicity)(The on-target toxicities result from the inability of engineered T cells to distinguish between normal cells and cancer cells that express the targeted Ag.)

Cytokine release syndrome

Tumor lysis syndrome

Neurologic toxicities

Toxicities

Toxicities

On target/off tumor toxicities

Metastatic colon cancer patient died after 5 days of infusion of ERBB2+CAR-T cells

Low levels of ERBB2 express on lung epithelium (lung tox)

Renal cell carcinoma: 5/11 patients developed liver toxicity

Cytokine syndrome

Elevated levels of pro-inflammatory cytokines

Treatable by anti-IL-6mAb and steroids

Challenges of CAR-T cells

Efficacy & Long-term persistenceSubtypes of CD4+T cells (Th1, Th2, Th17, Th9 cells),

CD8+T cells

naïve, central memory; long-term

effector; active but short lived

Patient conditioning before ACTReduced-intensity or non-myeloablative

Increased intensity myelo ablative

Tumor targetTarget antigen is critical determinant for efficacy & safety

Ideal target uniquely express on tumor cells or on cells which are not essential for survival

Trafficking of CAR T cells to tumorExpression of addressins

Route of CAR-T cell infusion

Intra-tumoral/intravenous

Optimal co-stimulation of T cells

Determinants of successful ACT: CAR-T cells

CART Programs at Academic Centers

Kenderian et al. BBMT in press.

Moving Forward with CAR-T Cells

Development of CAR modified T cellcancer therapy

• CAR design• CAR modified T cell trafficking• CAR modified T cell persistence• CAR modified T cell perseverance• Conditioning regimen including

Lymphodepletion

Moving Forward: Armored CARs

Moving Forward

New CAR T cell Strategies• Combination with immune

stimulator drugs• Different CAR T cell populations

• Cytotoxic T-cell (CD8)

• Helper T-cell (CD4)

• Regulatory/suppressor T-cell

• Memory T-cell

Classfication of T-cell

IL23R

Adoptive T cell therapy: Right T cell population?

CD4+ T cells

Engineering Th9 cells: TAA specific Tumor model

OT2 TCR transgenic mice (CD4 cells recognize ova)

B16F10-Ova cells Follow tumor development

Normal WT or Rag1-/-

CD4+CD25-CD62L+

Th17

Th9TGFβ + IL-4

TGFβ + IL-6

Naïve OT2

Engineer tumor specific Th9 cells

Generation of Ovalbumin expressing B16 tumor cells(Lentiviral method)

Phase I Trial of Stem Cell Gene Therapy for HIV in AIDS Lymphoma Patients

Why Is It So Difficult to Cure HIV?

Microglial and Kupffer cells,

http://abcnews.go.com/Health/french-hiv-study-means-hiv-babys-cure/story?id=18741318#.UZFht7VOQrU

http://www.cnn.com/2013/03/18/health/hiv-functional-cure

Candidate Product

an shRNA against CCR5, Cell entry phase

a chimeric Trim5α: pre-integration stage by disrupting viral capsid uncoating

a TAR decoy: post-integration/pre-transcription

All transferred by a single self-inactivating lentiviral vector

Gene modified HSC with a triple combination of anti-HIV molecules:

Down regulation of CCR5 expression on 1TAX cells

pre-selective anti-HIV vectors

CD34+ HSCs

vector transduced HSCs

purification

purified HIV-resistant HSCs

transduction

Pre-selective Approach

Barclay et al. 2014 Stem Cells

Enrichment of 1TAX vector transduced cells

Phenotypic profile of 1TAX vector transduced peripheral blood T cells

HIV-1 challenge of 1TAX HSPC derived macrophages

Maintenance of human CD4+ T cells in vivo in 1TAX cell engrafted NRG mice infected with HIV-1

Study Identifier or Title

Study Objective(s) Test Article and Manufacturing Process Used

Animal Model and Number of Animals pergroup

Key Outcome(s)

Immunogenicity of chimeric T5A To determine any immunogenicity to chimeric T5A Research grade NA No reaction detected (t-test, p>0.05) In vitro toxicity To determine any toxicity in transduced PBMCs Research grade NA No toxicity detectedIn vitro CFU assay To determine any toxicity in transduced CD34+ HPCs Clinically equivalent NA No toxicity detected (t-test, p>0.05)Purification of transduced cells To determine the levels of enrichment of transduced HPC Clinically equivalent NA Purification of >95% of transduced cellsIn vitro CFU/IL2 assay To determine if the addition of IL2 increased transduced cell

proliferationClinically equivalent NA No increased proliferation (t-test, p>0.05)

Proto-oncogene QPCR To determine any upregulation of proto-oncogenes Research grade NA No increased expression (t-test, p>0.05)CD122/CD132 flow cytometry To determine any upregulation of CD122 or CD132 Clinically equivalent NA No upregulation of CD122 of CD132 (t-

test, p>0.05)In vitro immortalization To determine if immortalization occurs with transduced cells Clinically equivalent NA No immortalization had occurred

Phenotypic analysis of HPC derived macrophages To determine if transduced cells have a normal macrophage phenotype Clinically equivalent NA Transduced macrophages had a normal macrophage phenotype (t-test, p>0.05)

Anti-HIV gene expression To determine the expression of anti-HIV genes Clinically equivalent NA Detection of expression of all three genes

Stability of vector To determine the stability of the vector in transduced cells Clinically equivalent NA No deletions or rearrangements were detected

CCR5 downregulation To determine the levels of CCR5 downregulation Clinically equivalent NA >93% downregulation of CCR5 (t-test, p<0.05)

In vivo engraftment and multi-lineage hematopoiesis

To determine the in vivo engraftment and multi-lineage hematopoiesis levels of transduced cells

Clinically equivalent NRG (N=10) Normal engraftment levels were observed in the peripheral blood + lymphoid organs (t-test, p>0.05)

Tumorigenicity To determine the tumorigenicity of transduced cells Clinically equivalent NRG (N=8) No tumors were detectedCytokine secretion profile To determine if engrafted human T cells are functionally normal Research grade NRG (N=3) Normal cytokine profiles (t-test, p>0.05)

Karyotyping To determine if transduced cells have a normal karyotype Research grade NA A normal karyotype was observedSequencing To determine if the sequence of integrated vector is correct Clinically equivalent NA Correct sequence was observedSafety of rapamycin To determine the safety of rapamycin treated CD34+ HSC Clinically equivalent NA No toxicity observed (t-test, p>0.05)In vitro/ex vivo HIV-1 challenge To determine the levels of protection of transduced cells Clinically equivalent NA >3 logs inhibition of HIV-1 infection (t-

test, p<0.05)In vivo HIV-1 challenge To determine if transduced and engrafted human immune cells

inhibited HIV-1Clinically equivalent NRG (N=14) lower levels of HIV-1 viremia (t-test,

p<0.05)Escape mutants To determine if any viral escape mutants arose in HIV-1 challenge

experimentsResearch grade NA No escape mutants were detected

Selective survival To determine a selective survival advantage of transduced cells Clinically equivalent NRG (N=8) A selective survival advantage was detected with transduced cells (t-test, p<0.05)

HIV-1 integration To determine if HIV-1 challenge virus was able to create provirus Research grade NA No integration of HIV-1 was detected

CD4+ cell maintenance To determine if normal CD4+ cell levels could be maintained in transduced cell engrafted mice

Clinically equivalent NRG (N=14) Normal CD4+ cell levels were observed in mice engrafted with transduced cells (t-test, p<0.05)

Lentiviral transduction % To determine if rapamycin treatment increased vector transduction Clinically equivalent NA A 3-4 fold increase in transduction (t-test, p<0.05)

A Phase I Study of Stem Cell Gene Therapy for HIV Mediated by Lentivector Transduced, Pre-selected CD34+ Cells

NCI Protocol: AMC-097

Combinatorial anti-HIV lentivirus:• an shRNA against CCR5• a chimeric Trim5α• a TAR decoy• CD25 preselection of

Phase I Study of Anti-HIV Gene Modified Hematopoietic Stem cells Transplant in Patient With HIV Related

Lymphomas to Cure Both Their Lymphoma and HIV

Regulatory:NIH RAC and FDA IND approval have been obtainedFunding:NIH CTEP and CIRM

High-grade non-Hodgkin’s lymphoma, meeting one of the following criteria

• In partial remission,• Relapsed after initial complete remission,• Failed induction therapy, but responds to salvage therapy (i.e., chemosensitive disease),• In complete remission with high-risk features as specified by the International Prognostic Index.

Advanced stage follicular lymphoma, that have failed at least two lines of therapy multi-agent chemotherapy, but responds to salvage therapy (i.e., chemosensitive disease)

Advanced stage Mantle cell lymphoma with Ki-67 > 10% in first complete remission

Hodgkin’s lymphoma, meeting one of the following criteria

• In first, or greater relapse after initial complete remission,• In partial remission,• Failed induction therapy, but responds to salvage therapy (i.e., chemosensitive disease).

Burkitt’s lymphoma, meeting one of the following criteria:•In second complete remission after relapse following initial complete remission,•Failed induction therapy, but responds (very good partial remission, complete remission, or near complete remission) to salvage therapy (i.e., chemosensitive disease).

Plasmoblastic lymphomas, or peripheral T cell lymphoma (with the exception of ALK+ type in first or second complete remission) (timeline 4 months prior to start of trial).

The primary endpoint of the study is:

Safety, defined as timely engraftment (the collective establishment of a persistent absolute neutrophil count of at least 500 cells/mm3 and platelet count of 20,000 cells/mm3 without transfusion for 3 consecutive days) at one month post transplant, in the absence of any study candidate specific grade 3 and 4 non-hematopoietic organ toxicity or any clonal expansion;

Major Secondary Objectives

• Efficacy of the candidate product, defined as establishment of > 5% mononuclear blood cells expressing anti-HIV genes in the peripheral blood at 3 months post-transplant.

• To determine the presence, quantity, and duration of gene modified HIV-1 resistant peripheral blood cells and gut mucosal immune cells.

• To study the integration sites of vector sequences in circulating cells.• Minor Secondary Objectives• To study progression-free survival.• To study overall survival.• To study complete response rate and duration.• To study partial response rate and duration.• To study time to neutrophil engraftment (first of 3 consecutive days of ANC > 500

cells/mm3.• To study time to platelet engraftment (first of 3 consecutive days of platelets > 20,000

cells/mm3 without platelet transfusions 7 days prior).• To study hematologic function at Day 100 (ANC > 1500, Hb > 10g/dl without transfusion

and platelets > 100,000)• To study CD4 recovery at the conclusion of the trial.• To study safety in terms of toxicities, infections, transfusions, and infusion-related

reactions.• To study HIV-1 viral load over time.• To study persistence of vector-transduced cells over time.

Study Cohorts Ratio of Transduced versus Untransduced Stem Cells

1 1:1

2 5:1

3 1:0

Study Cohorts

Study Cohorts

Ratio of Transduced vs Un-transduced Stem

Cells

Minimum Number of Transduced/Un-transduced

Cells Acceptable. CD34 cells/kg

Goal for Number of Transduced Cells.

CD34 cells/kg

1 1:1 1 X 106:1 X 106 2-5 X 106: 2-5 X 106

2 5:1 2 X 106:1 X 106 5 X 106:1 X 106

3 1:0 2 X 106: 0 5-10 X 106: 0

How Many CD34 Do We Need?

• At or after six months post transplant• Voluntarily • Eight-week period• Only in the third cohort • CD4 count of 500 or higher with no detectable viral load by single copy PCR.

ART withdrawal

Immunology Study in HIV-Stem Cell Protocol

I. Reconstitution of Immune System in Peripheral Blood:1. Subsets and Differentiation of CD4 and CD8 T Lymphocytes:

Developed from our previous and current studies, flow cytometric assays will be used:

a. Determine the memory and naïve CD4 and CD8 T cells as well as the T cell activation[CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD69, CD71 and viable cell stain]

b. Determine the Treg, NK and NK-T cell subsets:[CD3, CD4, CD8, CD25, IL-7R, FoxP3, CD16, CD56 and viable cell stain]

*expression of homing molecules CD18, CD31, CD54 may be added to this test.

These assays will be performed after stem-cell transplant to monitor the re-establishment of T cell system.

2. Functional Responses Upon Reconstitution of T Cell ImmunityThe following studies are proposed, primarily to measure the cytokine responses:a. Total Th1/Th2/Th17 subsets will be measured after mitogen stimulation (1-2 months after transplant)

[CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2, IL4, IL10]

b. Antigen specific T cell responses will be measured after stimulations with CEF and HIV-gag peptides[proposed flow cytometric assay: CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2][alternative assay, a dual-color ELISpot Assay to evaluate IFNg/IL2 producing T cells, if PBMC cells are limited]

c. If adequate PBMC are collected, proliferation responses will be tested using CFSE assays

Immunology Study in HIV-Stem Cell Protocol

II. Reconstitution of T Cell Immune System in Gastrointestinal-associated lymphoid tissue:

1. After stem cell transplant (2-4 months?), GI mucosal biopsy samples will be obtained.2. Biopsy tissues digested with Collagenese will be used for phenotyping

and functional assay:a. Determine the memory and naïve CD4 and CD8 T cells as well as the

T cell activation[CD3, CD4, CD8, CCR7, CD45RA, CD38, HLA-DR, CD69, CD71 and

viable cell stain]b. Ag-specific T cell responses will be measured after mitogen, CEF

and HIV stimulations[CD3, CD4, CD8, and intra-cellular IFNg, TNFa, IL2, IL4, IL10]

[alternatively, ELISpot will be used to evaluate IFNg producing T cells]

AIDS Malignancy Consortium

Sponsored by NCI 25 US Sites 9 International Sites

initial CD34 %

total MNCsb

initial CD34+

isolated CD34+

transduced CD34+

CD34+post-transduction

CD34+post CD25 selection

% yield from initial CD34+

sample

chemoExpt 1 1.16 1.12x10^8 1.3x10^6 1.48x10^6 1.44x10^6 1.6x10^6 1.08x10^6 83.1 mobilized/lymphoma

G+Plerixafor/multipleExpt 2 0.35 4.3x10^8 1.5x10^6 1.1x10^6 1.1x10^6 1.07*10^6 1.84x10^6 122.7 myeloma

chemoExpt 3 1.11 8.32x10^7 923,520 1.02x10^6 1.5x10^6 1.16x10^6 620,000 67.1 mobilized/lymphoma

G+Plerixafor/multipleExpt 4 0.37 4.44x10^8 1.64x10^6 1.45x10^6 1.5x10^6 1.26x10^6 1.15x10^6 70.1 myeloma

Expt 5 4.8 5.12x10^7 2.46x10^6 2.13x10^6 2.35x10^6 1.875x10^6 1.3x10^6 52.8 cord blood

G+Plerixafor/multipleExpt 6 0.23 5.79x10^8 1.33x10^6 870,000 1.57x10^6 1.27x10^6 552,056 41.5 myeloma

average%CD34

recoveredc89.65

Experiments MNCreceived

Total number of CD34 cells received

Total number of CD34scollected from the CD34 column (percentage of cell recovery)

Total number of CD34s collected after CD25 column (percentage of cell recovery)

Purity for the CD25+cells

1 4.7x10^10 3.57x10^8 1.55x10^8 (43.4%) 3.3x10^6 (19%) 25.4%

2 (label check expt) 5.8x10^10 5.1x10^8 4.28x10^8 (83.9%) N/A N/A

3 5.8x10^10 1.18x10^9 9.20x10^8 (78.0%) 6.3x10^7 (16.7%) 97.4%

4 5.7x10^10 2.10x10^8 1.49x10^8 (71.0%) 3.65x10^7 (79.9%) 86.7%

CTS1-08289Patient #1

summary of cell processing andrelease tests

MB is a 50 years old WM with relapsed Hodgkin's lymphomaPatient had a very poor collection with G+Mozobil.Febrile syndrome after collection.Disease relapsed while we were waiting for the second round of collectionWent to remission with ARA-C+etoposidePoor collection after chemomobilization.Added mozobil and given bid G-CSF with improved collectionWe initially had a problem with high granulocyte content but troubleshooted around this problem. CD34+ recovery, purity and viability increased.Transductions were high enough, and end CD34+/CD25+ cell purity was good. However, CD34+/CD25+ recovery was again low.

Patient #1 Mobilized Leukopaks1932-1936 are patient leukopak ID#s obtained on consecutive days. Due to poor CD34+ purity,viability, and recovery, we did not use leukopaks 1932-1934 for further transduction andseparation.

Leukopak id# Initial

CD34%

Theoretical yield

Granulocyte %

Recovery

% CD34 purity

% recovered

% viable

changed protocol to include increased IVIG with apreincubation period prior to labeling

1932/1933 1934 1935 1936

0.21 0.28 0.30 0.41

2.9x10^8 2.5x10^8 3.7x10^8 5.81x10^8

42 53 84 46

4.9x10^7 1.27x10^8 2.9x10^8 2.11x10^8

65.74 48.0 82.2 90.5

11.2 24.3 64.4 32.9

81.79 69.82 96.6 98.5

1935:1936:

cells transduced = 2.6x10^8 cells transduced = 1.8x10^8

Transduction efficiency:1935 = 23.6%1936 = 31.2%

Total CD34+/CD25+ cells:1935 = 6.136x10^71936 = 5.616x10^7

Theoretical yield at 100% efficiency= 1.18x10^8CD34+/CD25+ cellsOnly need to recover 7.0x10^7 (59.3%) for the 99kg patient

Recovered ~2.5x10^7 CD34+/CD25+ cells

• Only used the cells from leukopaks 1935 and 1936 dueto bad

purity and viability from others

How these genetic modification will affect hematopoietic cells

Immunological Reaction: elimination Differentiation issues Chemokine-cytokine-integrin disturbances

resulting in motility/homing/functional issues

ART Immunological Non-respondersFuture Directions

Very low doses of radiation can results in high level of engraftment

Principal InvestigatorAbedi

External Advisory BoardKohn, Rossi, Torbett,

Volberding

DNA monitoring

CoreAnderson

Regulatory LeadBauer

CMC (Bauer)• GMP facilityoVector productionoProgenitor LaboQA/ Control

CMC/Product Development Lead

Richman

Immunology Specialty

CorePollard

Clinical Project Manager TBD

Clinical LeadAbedi

HIV teamUC Davis

UCSF

Transplant team

UC DavisUCSF

FDA

Preclinical team Clinical team

Community AdvocateTBD

Recruitment

Co-PIAnderson

IRB

Preclinical Project Manager Bauer

Clinical Operations• CRC UCD (Quirch, Eddings)• CRC UCSF (Boyer, Murray,

Romano)• Data management (TBD)

Clinical Operations Core• CTSU• DSMB (TBD)• Biostatistics (Beckett)

Regulatory• FDA• NIH RAC• UC Davis Biosafety

Biologics Consulting Group, Inc

Miller

Acknowledgement